Compositions and Methods for Inhibiting Gene Expression of Hif2alpha

ABSTRACT

RNA interference (RNAi) triggers and RNAi trigger conjugates for inhibiting the expression of Hif2α (EPAS1) gene are described. Pharmaceutical compositions comprising one or more Hif2α RNAi triggers optionally with one or more additional therapeutics are also described. Delivery of the described Hif2α RNAi triggers to tumor cells in vivo provides for inhibition of Hif2α gene expression and treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/954,316, filed Apr. 16, 2018 which is a divisional of U.S. patentapplication Ser. No. 15/166,311, filed May 27, 2016 (now U.S. Pat. No.9,976,141), which claims benefit to U.S. Provisional Patent ApplicationSer. No. 62/168,244 filed on May 29, 2015, the disclosure of each ofwhich is specifically incorporated by reference herein.

BACKGROUND

EPAS1 is a member of the HIF (hypoxia inducible factor) gene family.Also known as Hif2alpha or Hif2α, EPAS1 encodes half of a transcriptionfactor involved in the induction of genes regulated by oxygen, and whichis induced as oxygen levels fall (a condition known as hypoxia).

Certain variants of this gene provide protection for people living athigh altitude. However, at low altitude, over-expression of wild-type(WT) EPAS1 is associated with increased hypertension and stroke, andwith symptoms similar to mountain sickness. Mutations in this gene areassociated with erythrocytosis familial type 4 and pulmonaryhypertension. EPAS1 can cause excessive production of red blood cells,leading to inhibited reproductive abilities or even death.

EPAS1 has been shown to be required for expression of, or enhance theexpression of, various genes involved in an assortment of diseases,including tumor progression. For example, EPAS1 may play a role in theprogression of uveal melanomas, possibly by promoting the autocrine loopVEGF-pVEGFR2/KDR, and by enhancing the expression of LDHA, thusconferring a growth advantage.

EPAS1 has also been shown to be involved in, or upregulates expressionof, other factors, including: cMyc (which favors cell proliferation,transformation, neoplasia and tumorigenesis, and which is highlyexpressed in most cancers); Interleukin 8 (a pro-inflammatory mediator,e.g., in gingivitis and psoriasis); SP-1 (a transcription factorinvolved in IL-8 regulation and a coactivator of cMyc); LDH5 (which islinked with tumor necrosis and increased tumor size); and LANA (LatencyAssociated Nuclear Antigen, which is associated with Kaposi'ssarcoma-associated Herpesvirus). In addition, HIF (hypoxia inducedfactor) activity may play a role in angiogenesis required for cancertumor growth. EPAS 1 may also be involved in several other diseases,including inflammation, chronic inflammation, neovascular diseases,rheumatoid arthritis, renal cancer, clear cell renal cell carcinoma (andmetastases of this and other cancers), melanoma, uveal melanoma,chondrosarcoma, and multiple myeloma.

Mutations in EPAS1 gene have been correlated to early onset ofneuroendocrine tumors such as paragangliomas, somatostatinomas and/orpheochromocytomas. The mutations are commonly somatic missense mutationslocated in the primary hydroxylation site of HIF-2a.

These mutations are believed to disrupt the proteinhydroxylation/degradation mechanism and lead to protein stabilizationand pseudohypoxic signaling. In addition, neuroendocrine tumors releaseerythropoietin (EPO) into circulating blood, and lead to polycythemia.

SUMMARY

Described herein are Hif2α (also termed EPAS, or Hif2alpha)gene-specific RNA interference (RNAi) trigger molecules (also termedRNAi agent, RNAi trigger, or trigger) able to selectively andefficiently decrease expression of Hif2α. Each RNAi trigger includes atleast a sense strand and an antisense strand. The sense strand and theantisense strand can be partially, substantially, or fully complementaryto each other. The length of the RNAi trigger sense and antisensestrands described herein each can be 16 to 30 nucleotides in length. Insome embodiments, the sense and antisense strands are independently 17to 26 nucleotides in length. The sense and antisense strands can beeither the same length or different lengths. The RNAi triggers describedherein, upon delivery to a cell expressing the Hif2α gene, inhibit theexpression of the Hif2α gene in vitro or in vivo. Examples of Hif2α RNAitrigger sense strands and antisense strands that can be used in a Hif2αRNAi trigger are provided in Tables 1-2 and 5.

A sense strand of an Hif2α RNAi trigger contains a nucleotide sequencehaving at least 90% identity over a core stretch of at least 16consecutive nucleotides to a sequence in an Hif2α mRNA. In someembodiments, the nucleotide sequence having at least 90% identity to asequence in the Hif2α mRNA is 16, 17, 18, 19, 20, 21, 22, or 23nucleotides in length. An antisense strand of an Hif2α RNAi triggercontains a nucleotide sequence having at least 90% complementary over acore stretch of at least 16 consecutive nucleotides to a sequence in theHif2α mRNA and the corresponding sense strand. In some embodiments, thenucleotide sequence having at least 90% complementarity to a sequence inthe Hif2α mRNA or the corresponding sense strand is 16, 17, 18, 19, 20,21, 22, or 23 nucleotides in length.

In some embodiments, one or more Hif2α RNAi triggers are delivered totarget cells or tissues using any oligonucleotide delivery technologyknown in the art. Nucleic acid delivery methods include, but are notlimited to, by encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, proteinaceousvectors or Dynamic Polyconjugates™ (DPCs). In some embodiments, an Hif2αRNAi trigger is conjugated to a targeting group, such as aintegrin-binding compound. In some embodiments, an Hif2 RNAi trigger isconjugated to a delivery polymer or vehicle. The delivery polymer can bea reversibly modified membrane active polyamine. The delivery polymercan also be an integrin-targeted reversibly modified membrane activepolyamine.

An integrin-targeted reversibly modified membrane active polyaminecomprises a membrane active polyamine conjugated to one or moreintegrin-binding compounds via reversible physiologically labilecovalent linkages. In some embodiments, the integrin targeted reversiblymodified membrane active polyamine further comprises the membrane activepolyamine conjugated to one or more steric stabilizers via reversiblephysiologically labile covalent linkages. Integrin-binding compounds canbe, but are not limited to, RGD peptides and RGD mimics. Reversiblephysiologically labile covalent linkages include, but are not limitedto, dipeptide amidobenzyl carbamate linkages, tetrapeptide linkages, anddisubstituted maleamate linkages.

The Hif2α RNAi triggers are optionally combined with one or moreadditional (i.e., second, third, etc.) therapeutics. A secondtherapeutic can be another Hif2α RNAi trigger (e.g., a Hif2α RNAitrigger which targets a different sequence within the Hif2α target). Anadditional therapeutic can also be a small molecule drug, antibody,antibody fragment, and/or vaccine. The Hif2α RNAi triggers, with orwithout the one or more additional therapeutics, can be combined withone or more excipients to form pharmaceutical compositions.

The present disclosure also encompasses methods of treating a humansubject having a pathological state mediated at least in part by Hif2αexpression, the methods comprising the step(s) of administering to thesubject a therapeutically effective amount of an Hif2α RNAi trigger orHif2α RNAi trigger-containing composition. The method of treating asubject with an Hif2α RNAi trigger or Hif2α RNAi trigger-containingcomposition can optionally be combined with one or more steps ofadministering one or more additional (i.e., second) therapeutics ortreatments. The Hif2α RNAi trigger and additional therapeutics can beadministered in a single composition or they made be administeredseparately. Non-limited examples of additional therapeutics include, butare not limited to, VEGFR inhibitors (such as SUTENT®, NEXAVAR®,VOTRIENT®, AVASTIN®, INLYTA®, CABOZANTINIB®), Cytokines (such as IL-2,IFN-α), mTor inhibitors (such as EVEROLIMUS®, TEMSIROLIMUS®), anti-PD1drugs (such as OPDIVO® and KEYTRUDA®), anti-CTLA4 (such as YERVOY®),drugs targeting signal transduction pathway components in cancer cells(such as VEGF, PI-3-kinase, MEK, JAK, Akt, MYC, Met, Src-family kinases,Abl, Axl, Mer), anti-PD-L1, anti-PD-L2, anti-TIM3, anti-LAG3, anti-CD28,anti-OX40, anti-OX-40L, anti-CD39. anti-CD40, anti-CD80, anti-CD86,anti-CD137, anti-41BBL, anti-TIGIT, anti-GITR, anti-GIRTL, anti-CD155,anti-Fas, anti-FasL, anti-TRAIL/TRAIL-L, IDO-1 inhibitor, and TDO-2inhibitor.

The pharmaceutical compositions can be administered in a number of waysdepending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be topical (e.g., by atransdermal patch), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer, intratracheal, intranasal),epidermal, transdermal, oral or parenteral. Parenteral administrationincludes, but is not limited to, intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion;subdermal (e.g., via an implanted device), intracranial,intraparenchymal, intrathecal, and intraventricular, administration.

The described Hif2α RNAi triggers and/or compositions can be used inmethods for therapeutic treatment of diseases, including but not limitedto: cancer, renal cancer, clear cell renal cell carcinoma, non-smallcell lung cancer, astrocytoma (brain cancer), bladder cancer, breastcancer, chondrosarcoma, colorectal carcinoma, gastric carcinoma,glioblastoma, head and neck squamous cell carcinoma, hepatocellularcarcinoma, lung adenocarcinoma, neuroblastoma, melanoma, multiplemyeloma, ovarian cancer, rectal cancer, metastases, gingivitis,psoriasis, Kaposi's sarcoma-associated herpesvirus, preemclampsia,inflammation, chronic inflammation, neovascular diseases, and rheumatoidarthritis. Such methods comprise administration of an Hif2α RNAi triggeras described herein to a subject, e.g., a human or animal subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemical structures representing PEG_(n)-FCitFP-TFP modifyingagents.

FIG. 2. Chemical structures representing RGD-PEG_(n)-FCitFP-TFPmodifying agents.

FIG. 3. Chemical structures representing RGD-PEG_(n)-ACit-PABC-PNPmodifying agents.

FIG. 4. Chemical structures representing aldehyde-PEG_(n)-FCit-PABC-PNPmodifying agents.

FIG. 5. Chemical structures representing aldehyde-PEG_(n)-ACit-PABC-PNPmodifying agents.

FIG. 6. Chemical structures representing SPDP-PEG_(n)-FCit-PABC-PNPmodifying agents.

FIG. 7. Chemical structures representing PEG_(n)-ACit-PABC-PNP andPEG_(n)-FCit-PABC-PNP modifying agents.

FIG. 8. Graph illustrating serum SEAP levels during treatment in mice.Fold-changed in serum SEAP levels relative to day (−1) pre-dose levels.For G1 and G2, n=4. For G3, n=3.

FIG. 9. Tumor gross morphology after 3 weekly treatments in mice. G1,vehicle; G2 400 μg Hif2α-ITG-DPC; G3 280 μg Hif2α-ITG-DPC. Both kidneysfrom each animal are shown. Tumor was implanted into the kidney shown onthe right. Tumor from treatment groups were all significantly smallerand showed some discoloration when compared to controls.

FIG. 10. H&E staining of tumor formalin fixed paraffin sections. Thinarrows indicate apoptotic cells. Thick arrows indicate macrophageinfiltration. Panel A, G1 vehicle treated. Typical tubular type RCCmorphology with rare apoptotic cells (thin arrows). Panel B, G2 400 μgHif2α-ITG-DPC treated. Massive necrotic center with numerous apoptoticcells in surrounding areas and overall loss of tumor structure. Panel C,G3 280 μg Hif2α-ITG-DPC treated. Destruction of typical tubular tumorstructures with macrophage infiltration and numerous apoptotic cells.

DETAILED DESCRIPTION

Described herein are RNAi triggers for inhibiting expression of theHif2α gene (referred to herein as Hif2α RNAi triggers). Each Hif2α RNAitrigger comprises a sense strand and an antisense strand. The sensestrand and the antisense strand are partially, substantially, or fullycomplementary to each other. In some embodiments, the length of theherein described RNAi trigger sense and antisense strands areindependently 16 to 30 nucleotides in length. In some embodiments, thelength of the herein described RNAi trigger sense and antisense strandsare independently 17 to 26 nucleotides in length. In some embodiments,the herein described RNAi trigger sense and antisense strands areindependently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides inlength. The sense and antisense strands can be either the same length orthey can be different lengths. In some embodiments, the sense strand isabout 19 nucleotides in length while the antisense strand is about 21nucleotides in length. In some embodiments, the sense strand is about 21nucleotides in length while the antisense strand is about 23 nucleotidesin length. In other embodiments, the sense and antisense strands areindependently 17-21 nucleotides in length. In some embodiments, both thesense and antisense strands are each 21-26 nucleotides in length.Examples of nucleotide sequences used in forming Hif2α RNAi triggermolecules are provided in Tables 1-2 and 5.

RNAi triggers include, but are not limited to: short interfering RNAs(siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpinRNAs (shRNA), and dicer substrates (U.S. Pat. No. 8,084,599 8,349,809and 8,513,207). The RNAi triggers described herein, upon delivery to acell expressing the Hif2α gene, inhibit or knockdown expression of Hif2αgene in vitro or in vivo through the biological process of RNAinterference (RNAi).

An Hif2α RNAi trigger comprises a sense strand and an antisense strandeach containing a core sequence of 16-23 nucleobases in length. Anantisense strand core sequence is 100% (perfectly) complementary or atleast 90% (substantially) complementary to a nucleotide sequence(sometimes referred to, e.g. as a target sequence) present in the Hif2αmRNA. A sense strand core sequence is 100% (perfectly) complementary orat least 90% (substantially) complementary to a sequence in theantisense strand and thus the sense strand core sequence is perfectlyidentical or at least 90% identical to a nucleotide sequence (targetsequence) present in the Hif2α mRNA. A sense strand core sequence can bethe same length as a corresponding antisense core sequence or it can bea different length. In some embodiments, the antisense strand coresequence is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. Insome embodiments, the sense strand core sequence is 16, 17, 18, 19, 20,21, 22, or 23 nucleotides in length.

The Hif2α RNAi trigger sense and antisense strands typically anneal toform a duplex. Within the complementary duplex region, the sense strandcore sequence is at least 90% complementary or 100% complementary to theantisense core sequence. In some embodiments, the sense strand coresequence contains a sequence of at least 16, at least 17, at least 18,at least 19, at least 20, or at least 21 nucleotides that is at least90% or 100% complementary to a corresponding 16, 17, 18, 19, 20, or 21nucleotide sequence of the antisense strand core sequence (i.e., thesense strand and antisense core sequences of an Hif2α RNAi trigger havea region of at least 16, at least 17, at least 18, at least 19, at least20, or at least 21 nucleotides that is at least 90% base paired or 100%base paired.)

As used herein, the term “sequence” or “nucleotide sequence” refers to asuccession or order of nucleobases, nucleotides, and/or nucleosides,described with a succession of letters using the standard nucleotidenomenclature and the key for modified nucleotides described herein.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence(e.g., RNAi trigger sense strand or Hif2α mRNA) in relation to a secondnucleotide sequence (e.g., RNAi trigger antisense strand), refers to theability of an oligonucleotide or polynucleotide comprising the firstnucleotide sequence to hybridize (form base pair hydrogen bonds) andform a duplex or double helical structure under certain conditions withan oligonucleotide or polynucleotide comprising the second nucleotidesequence. Complementary sequences include Watson-Crick base pairs ornon-Watson-Crick base pairs and include natural or modified nucleotidesor nucleotide mimics as long as the above requirements with respect totheir ability to hybridize are fulfilled. “Perfectly complementary” or“fully complementary” means that all (100%) of the bases in a contiguoussequence of a first polynucleotide will hybridize with the same numberof bases in a contiguous sequence of a second polynucleotide. Thecontiguous sequence may comprise all or a part of a first or secondnucleotide sequence. As used herein, “partial complementary” means thatin a hybridized pair of nucleobase sequences, at least 70% of the basesin a contiguous sequence of a first polynucleotide will hybridize withthe same number of bases in a contiguous sequence of a secondpolynucleotide. As used herein, “substantial complementary” means thatin a hybridized pair of nucleobase sequences, at least 85% of the basesin a contiguous sequence of a first polynucleotide will hybridize withthe same number of bases in a contiguous sequence of a secondpolynucleotide. The terms “complementary”, “fully complementary” and“substantially complementary” as used herein may be used with respect tothe base matching between the sense strand and the antisense strand ofan RNAi trigger, or between the antisense strand of an RNAi trigger anda sequence of an Hif2α mRNA. Sequence identity or complementarity isindependent of modification. For the purposes of determining identity orcomplementarity, for example, a and Af are complementary to U (or T) andidentical to A.

The sense strand and/or the antisense strand may optionally andindependently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides(extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of thecore sequences. The antisense strand additional nucleotides, if present,may or may not be complementary to the corresponding sequence in theHif2α mRNA. The sense strand additional nucleotides, if present, may ormay not be identical to the corresponding sequence in the Hif2α mRNA.The antisense strand additional nucleotides, if present, may or may notbe complementary to the corresponding sense strand's additionalnucleotides, if present.

As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotidesat the 5′ and/or 3′ end of the sense strand core sequence and/orantisense strand core sequence. The extension nucleotides on a sensestrand may or may not be complementary to nucleotides, either coresequence nucleotides or extension nucleotides, in the correspondingantisense strand. Conversely, the extension nucleotides on an antisensestrand may or may not be complementary to nucleotides, either coresequence nucleotides or extension nucleotides, in the correspondingsense strand. In some embodiments, both the sense strand and theantisense strand of an RNAi trigger contain 3′ and 5′ extensions. Insome embodiments, one or more of the 3′ extension nucleotides of onestrand base pairs with one or more 5′ extension nucleotides of the otherstrand. In other embodiments, one or more of 3′ extension nucleotides ofone strand do not base pair with one or more 5′ extension nucleotides ofthe other strand. In some embodiments, an Hif2α RNAi trigger has anantisense strand having a 3′ extension and a sense strand having a 5′extension.

In some embodiments an Hif2α RNAi trigger molecule comprises anantisense strand having a 3′ extension of 1, 2, 3, 4, 5, or 6nucleotides in length. In other embodiments, an Hif2α RNAi triggermolecule comprises an antisense strand having a 3′ extension of 1, 2, or3 nucleotides in length. In some embodiments, one or more of theantisense strand extension nucleotides comprise uracil or thymidinenucleotides or nucleotides which are complementary to the correspondingHif2α mRNA sequence. In some embodiments, the antisense strand extensioncan be, but is not limited to: uAu, uGu, udTsdT, usdTsdT, UfAu, Aua,Afsusa, UAU, uAfu, uau, udAu, uscu, usgu, uscsu, cAu, aUa, aua,u(invdA)u, cag, agu, gcg, caa, usasu, uAMTM, or usTMsAM (each listed 5′to 3′, notation is the same as for Table 2).

In some embodiments, an Hif2α RNAi trigger molecule comprises anantisense strand having a 5′ extension of 1, 2, 3, 4, or 5 nucleotidesin length. In other embodiments, an Hif2α RNAi trigger moleculecomprises an antisense strand having a 5′ extension of 1 or 2nucleotides in length. In some embodiments, one or more of the antisensestrand extension nucleotides comprises uracil or thymidine nucleotidesor nucleotides which are complementary to the corresponding Hif2α mRNAsequence. In some embodiments, the antisense strand extension includesor consists of dA, dT, pdT, vpdT, or u, wherein dA and dT representdeoxyadenosine and deoxythimidine nucleotides respectively. pdTrepresents a deoxythimidine nucleotide having a 5′ phosphate, vpdTrepresents a vinylphosphonate deoxythimidine nucleotide, and urepresents a 2′-OMe modified uracil nucleotide. An antisense strand mayhave any of the 3′ extensions described above in combination with any ofthe 5′ antisense strand extensions described, if present.

In some embodiments, an Hif2α RNAi trigger molecule comprises a sensestrand having a 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length.In some embodiments, one or more of the sense strand extensionnucleotides comprises adenosine, uracil, or thymidine nucleotides, ATdinucleotide, or nucleotides which correspond to nucleotides in theHif2α mRNA sequence. In some embodiments, the 3′ sense strand extensionincludes or consists of Af, invdA, invdT, A(invdT), Af(invdT), U(invdT),Uf(invdT), AfAbuAu, dTdT, or dTsdT, wherein Af and Uf represent2′-fluoro adenosine and uracil nucleotides respectively, invdA and invdTrepresent 3′-3′ linked (inverted) deoxyadenosine and deoxythimidinenucleotides respectively, Ab represents an abasic ribose, u represents a2′-OMe modified uracil nucleotide, dT represents a deoxythimidinenucleotide, sdT represents a deoxythimidine nucleotide having a 5′phosphorothioate, and U and A represent uracil and adenosineribonucleotides.

In some embodiments, an Hif2α RNAi trigger molecule comprises a sensestrand having a 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides inlength. In some embodiments, one or more of the sense strand extensionnucleotides comprise uracil or adenosine nucleotides or nucleotideswhich correspond to nucleotides in the Hif2α mRNA sequence. In someembodiments, the sense strand 5′ extension can be, but is not limitedto: uAuAus, uAuAu, UAUUAGfs, UfaUfaA, uauaA, AUAUU, AfuAfuU, auauU,uaUfau, uAuA(U_(UNA)), uauau, udAudAu, uuAga, uuAuu, uuGAu, uuaga,uAuga, aUaGas, uauaus, uAuaas, udAuau, adTaga, auaga, u(invdA)uau,gacau, ugaau, gcgau, uauga, uugga, or auaga (each listed 5′ to 3′.notation is the same as for Table 2). A sense strand may have a 3′extension and/or a 5′ extension.

Unmodified Hif2α RNAi trigger sense strand and antisense strandsequences are provided in Tables 1 and Table 5. In forming Hif2α RNAitriggers, each of the nucleotides in each of the sequences listed inTables 1 and 5 may be a modified nucleotide.

TABLE 1 Unmodified Hif2α RNAi trigger antisense strandand sense strand sequences. SEQ SEQ ID Antisense Strand ID Sense StrandNO. Sequence 5′ → 3′ NO. Sequence 5′ → 3′  1 AGUAAAACAAUUGUGUACUUU 57AGUACACAAUUGUUUUACUT  2 AGUAAAACAAUUGUGUACUUU 44 AAAGUACACAAUUGUUUUAC AAT  3 AUUCAUGAAAUCGUUACGUTG 52 ACGUAACGAUUUCAUGAATT  4AUUCAUGAAAUCGUUACGUUG 52 ACGUAACGAUUUCAUGAATT  4 AUUCAUGAAAUCGUUACGUUG53 ACGUAACGAUUUCAUGAAU  4 AUUCAUGAAAUCGUUACGUUG 54 ACGUAACGAUUUCAUGAAUT 4 AUUCAUGAAAUCGUUACGUUG 73 UAUACGUAACGAUUUCAUGA AUT  4AUUCAUGAAAUCGUUACGUUG 74 UAUACGUAACGAUUUCAUGA AUTT  5AUUCAUGAAAUCGUUACGUUG 47 AACGUAACGAUUUCAUGAAU AT T  6AUUCAUGAAAUCGUUACGUUG 77 UAUCAACGUAACGAUUUCAU GC GAAUTT  7TAAAUCGUUACGUUGACAGTT 67 CUGUCAACGUAACGAUUUAT  8 TAACCACAUACGUUGGAGUTT55 ACUCCAACGUAUGUGGUUAT  9 TAAGUUAAGCUCCCAUACATT 81 UGUAUGGGAGCUUAACUUAT10 TAAUCGUUACGUUGACAGGTT 63 CCUGUCAACGUAACGAUUAT 11TACGUUGACAGGUAGGGUUTT 45 AACCCUACCUGUCAACGUAT 12 TAGAGGAGCUUGUGUGUUCTT68 GAACACACAAGCUCCUCUAT 13 TAGCUUGUGUGUUCGCAGGTT 62 CCUGCGAACACACAAGCUAT14 TAGGAGCUUGUGUGUUCGCTT 69 GCGAACACACAAGCUCCUAT 15TAUCGUUACGUUGACAGGUTT 49 ACCUGUCAACGUAACGUAAT 15 TAUCGUUACGUUGACAGGUTT71 UAUACCUGUCAACGUAACGU AAT 16 TCAUGAAAUCGUUACGUUGTT 60CAACGUAACGAUUUCAUGAT 17 TCGUUACGUUGACAGGUAGTT 65 CUACCUGUCAACGUAACGAT 18TCUAGCAACAAAACCUUAATT 82 UUAAGGUUUUGUUGCUAGAT 19 TGAGCUUGUGUGUUCGCAGTT66 CUGCGAACACACAAGCUCAT 19 TGAGCUUGUGUGUUCGCAGTT 68 GAACACACAAGCUCCUCUAT20 TGAGGAGCUUGUGUGUUCGTT 64 CGAACACACAAGCUCCUCAT 21TGGAGCUUGUGUGUUCGCATT 79 UGCGAACACACAAGCUCCAT 22 TGGUACUGGGUGGCGUAGCTT70 GCUACGCCACCCAGUACCAT 23 TGUAAAACAAUUGUGUACUTT 56 AGUACACAAUUGUUUUACAT24 TUACGUUGACAGGUAGGGUTT 48 ACCCUACCUGUCAAGGUAAT 25TUCGUUACGUUGACAGGUATT 78 UCACUGUCAACGUAACGAAT 26 TUGAUAAACACUUAACCCATT80 UGGGUUAAGUGUUUAUCAAT 27 TUGUCACGAUGCGGUGGUUTT 61 CAGUGCAACGCCACCCAGAT28 TUUCAUGAAAUCGUUACGUCG 76 UAUAUCGACGUAACGAUUUC GCUAU AUGAAA 29TUUCAUGAAAUCGUUACGUCG 76 UAUAUCGACGUAACGAUUUC GCUGU AUGAAA 30TUUCAUGAAAUCGUUACGUTT 50 ACGUAACGAUUUCAUGAAA 30 TUUCAUGAAAUCGUUACGUTT 51ACGUAACGAUUUCAUGAAAT 30 TUUCAUGAAAUCGUUACGUTT 72 UAUACGUAACGAUUUCAUGAAAT 31 TUUCAUGAAAUCGUUACGUUG 59 CAACGUAACGAUUUCAUGAA GC A 32TUUCAUGAAAUCGUUACGUUG 75 UAUAUCAACGUAACGAUUUC GCUAU AUGAAA 33TUUCAUGAAAUCGUUACGUUG 75 UAUAUCAACGUAACGAUUUC GCUGU AUGAAA 34TUUCAUGAAAUCGUUACGUUG 75 UAUAUCAACGUAACGAUUUC GCUTT AUGAAA 35UCAUGAAAUCGUUACGUUGTT 58 CAACGUAACGAUUUCAUGA 35 UCAUGAAAUCGUUACGUUGTT 58CAACGUAACGAUUUCAUGA 36 UCUAGCAACAAAACCUUAATT 82 UUAAGGUUUUGUUGCUAGAT 37UGUAAAACAAUUGUGUACUTT 56 AGUACACAAUUGUUUUACAT 38 UGUAAAACAAUUGUGUACUUU56 AGUACACAAUUGUUUUACAT 39 UGUAAAACAAUUGUGUACUUU 43 AAAGUACACAAUUGUUUUACAA A 40 UUUCAUGAAAUCGUUACGUTT 51 ACGUAACGAUUUCAUGAAAT 40UUUCAUGAAAUCGUUACGUTT 72 UAUACGUAACGAUUUCAUGA AAT 41UUUCAUGAAAUCGUUACGUUG 50 ACGUAACGAUUUCAUGAAA 41 UUUCAUGAAAUCGUUACGUUG 51ACGUAACGAUUUCAUGAAAT 41 UUUCAUGAAAUCGUUACGUUG 72 UAUACGUAACGAUUUCAUGAAAT 42 UUUCAUGAAAUCGUUACGUUG 46 AACGUAACGAUUUCAUGAAA AT T

The Hif2α RNAi triggers described herein are formed by annealing anantisense strand with a sense strand. In some embodiments, an Hif2α RNAitrigger antisense strand comprises a nucleotide sequence of any of thesequences in Tables 1 and 5. In some embodiments, an Hif2α RNAi triggerantisense strand comprises the sequence of nucleotides 1-17, 2-17, 1-18,2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24,2-24, 1-25, 2-25, 1-26, or 2-26 of any of the sequences in Tables 1 and5. In some embodiments, an Hif2α RNAi trigger sense strand comprises thenucleotide sequence of any of the sequences in Tables 1 and 5. In someembodiments, an Hif2α RNAi trigger sense strand comprises the sequenceof nucleotides 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21,2-21, 1-22, 2-22, 1-23, 2-23, 1-24, 2-24, 1-25, 2-25, 1-26, or 2-26 ofany of the sequences in Tables 1 and 5.

In some embodiments, the sense and antisense strands of the RNAitriggers described herein contain the same number of nucleotides. Insome embodiments the sense and antisense strands of the RNAi triggersdescribed herein contain different numbers of nucleotides. In someembodiments, the sense strand 5′ end and the antisense strand 3′ end ofan RNAi trigger form a blunt end. In some embodiments, the sense strand3′ end and the antisense strand 5′ end of an RNAi trigger form a bluntend. In some embodiments, both ends of an RNAi trigger form a blunt end.In some embodiments, neither end of an RNAi trigger is blunt-ended. Asused herein a blunt end refers to an end of a double stranded triggermolecule in which the terminal nucleotides of the two annealed strandsare complementary (form a complementary base-pair).

In some embodiments, the sense strand 5′ end and the antisense strand 3′end of an RNAi trigger form a frayed end. In some embodiments, the sensestrand 3′ end and the antisense strand 5′ end of an RNAi trigger form afrayed end. In some embodiments, both ends of an RNAi trigger form afrayed end. In some embodiments, neither end of an RNAi trigger is afrayed end. As used herein a frayed end refers to an end of a doublestranded trigger molecule in which the terminal nucleotides of the twoannealed strands from a pair (i.e. do not form an overhang) but are notcomplementary (i.e. form a non-complementary pair). As used herein, anoverhang is a stretch of one or more unpaired nucleotides at the end ofone strand of a double stranded RNAi trigger molecule. The unpairednucleotides may be on the sense strand or the antisense strand, creatingeither 3′ or 5′ overhangs. In some embodiments the RNAi trigger moleculecontains: a blunt end and a frayed end, a blunt end and 5′ overhang end,a blunt end and a 3′ overhang end, a frayed end and a 5′ overhand end, afrayed end and a 3′ overhang end, two 5′ overhang ends, two 3′ overhangends, a 5′ overhang end and a 3′ overhand end, two frayed ends, or twoblunt ends.

A nucleotide base (or nucleobase) is a heterocyclic pyrimidine or purinecompound which is a constituent of all nucleic acids and includesadenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Asused herein, “G”, “g”, “C”, “c”, “A”, “a”, “U”, “u”, and “T”, eachgenerally stand for a nucleobase, nucleoside, nucleotide or nucleotidemimic that contains guanine, cytosine, adenine, uracil and thymidine asa base. Also as used herein, the term “nucleotide” can include amodified nucleotide or nucleotide mimic, abasic site, or a surrogatereplacement moiety.

As used herein, a “modified nucleotide” is a nucleotide other than aribonucleotide (2′-hydroxyl nucleotide). In some embodiments, an Hif2αRNAi trigger contains one or more modified nucleotides. In someembodiments, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or 100% of the nucleotides are modified.Modified nucleotides include, but are not limited to, deoxynucleotides,nucleotide mimics, abasic nucleotides (represented herein as X or Ab),2′-modified nucleotides, 3′ to 3′ linkages (inverted)nucleotides(represented herein as invdN, invN, invn, invX), non-naturalbase-comprising nucleotides, bridged nucleotides, peptide nucleic acids,2′,3′-seco nucleotide mimics (unlocked nucleobase analogues, representedherein as N_(UNA) or NUNA), locked nucleotides (represented herein asN_(LNA) or NLNA), 3′-O-Methoxy (2′ internucleotide linked) nucleotides(represented herein as 3′-OMen), 2′-F-Arabino nucleotides (representedherein as NfANA or Nf_(ANA)), morpholino nucleotides, vinyl phosphonatedeoxyribonucleotides (represented herein as vpdN), and vinyl phosphonatenucleotides. 2′-modified nucleotides (i.e. a nucleotide with a groupother than a hydroxyl group at the 2′ position of the five-memberedsugar ring) include, but are not limited to, 2′-O-methyl nucleotides(represented herein as a lower case letter ‘n’ in a nucleotidesequence), 2′-deoxy-2′-fluoro nucleotides (represented herein as Nf,also represented herein as 2′-fluoro nucleotide), 2′-deoxy nucleotides(represented herein as dN), 2′-methoxyethyl(2′-O-2-methoxylethyl)nucleotides (represented herein as NM or 2′-MOE),2′-amino nucleotides, 2′-alkyl nucleotides. It is not necessary for allpositions in a given compound to be uniformly modified. Conversely, morethan one modification may be incorporated in a single Hif2M RNAi triggeror even in a single nucleotide thereof. The Hif2α RNAi trigger sensestrands and antisense strands may be synthesized and/or modified bymethods known in the art. Modification at one nucleotide is independentof modification of another nucleotide.

Modified nucleotides also include nucleotides having modifiednucleobases. Modified nucleobases include, but are not limited to,synthetic and natural nucleobases, 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

In some embodiments 20% or fewer of the modified nucleotides are2′-fluoro modified nucleotides. In some embodiments, an Hif2α RNAitrigger sense strand contains a 2′-F nucleotide at position 11 from the3′ end. In some embodiments, an Hif2α RNAi trigger sense strand containsa 2′-F nucleotide at position 12 from the 3′ end. In some embodiments,an Hif2α RNAi trigger sense strand contains a 2′-F nucleotide atposition 13 from the 3′ end. In some embodiments, an Hif2α RNAi triggersense strand contains at least two 2′-F nucleotides at positions 11, 12,and 13 from the 3′ end. In some embodiments, an Hif2α RNAi trigger sensestrand contains 2′-F nucleotides at positions 11 and 12, positions 11and 13, or positions 12 and 13 from the 3′ end. In some embodiments, anHif2α RNAi trigger sense strand contains 2′-F nucleotides at positions11, 12, and 13 from the 3′ end.

In some embodiments, an Hif2α RNAi trigger antisense strand contains a2′-F nucleotide at position 2 from the 5′ end. In some embodiments, anHif2α RNAi trigger antisense strand contains a 2′-F nucleotide atposition 14 from the 5′ end. In some embodiments, an Hif2α RNAi triggerantisense strand contains 2′-F nucleotides at positions 2 and 14 fromthe 5′ end. In some embodiments, an Hif2α RNAi trigger contains at leasttwo 2′-F nucleotides at positions 11, 12, and 13 from the 3′ end of thesense strand and at positions 2 and 14 from the 5′ end of the antisensestrand.

In some embodiments, an Hif2α RNAi trigger antisense strand contains a2′-F nucleotide at position 4 from the 5′ end. In some embodiments, anHif2α RNAi trigger antisense strand contains a 2′-F nucleotide atposition 6 from the 5′ end. In some embodiments, an Hif2α RNAi triggerantisense strand contains a 2′-F nucleotide at position 8 from the 5′end. In some embodiments, an Hif2α RNAi trigger antisense strandcontains a 2′-F nucleotide at position 10 from the 5′ end. In someembodiments, an Hif2α RNAi trigger antisense strand contains a 2′-Fnucleotide at position 12 from the 5′ end. In some embodiments, an Hif2αRNAi trigger antisense strand contains at least two 2′-F nucleotides atpositions 4, 6, 8, 10, and 12 from the 5′ end. In some embodiments, anHif2α RNAi trigger antisense strand contains 2′-F nucleotides atpositions 4 and 6, positions 4 and 8, positions 4 and 10, positions 4and 12, positions 6 and 8, positions 6 and 10, positions 6 and 12,positions 8 and 10, positions 8 and 12, or positions 10 and 12 from the5′ end. In some embodiments, an Hif2α RNAi trigger antisense strandcontains at three 2′-F nucleotides at positions 4, 6, 8, 10, and 12 fromthe 5′ end. In some embodiments, an Hif2α RNAi trigger antisense strandcontains at least four 2′-F nucleotides at positions 4, 6, 8, 10, and 12from the 5′ end. In some embodiments, an Hif2α RNAi trigger antisensestrand contains 2′-F nucleotides at positions 4, 6, 8, and 10, positions4, 6, 8, and 12, positions 4, 6, 10, and 12, positions 4, 8, 10, and 12or positions 6, 8, 10, and 12 from the 5′ end.

In some embodiments, an Hif2α RNAi trigger antisense strand contains a2′-F nucleotide at position 2 and/or position 14 and one, two, or three2′-F nucleotides at positions 11, 12, and 13 from the 5′ end. In someembodiments, an Hif2α RNAi trigger contains a 2′-F nucleotide atposition 2 and/or position 14 and one, two, or three 2′-F nucleotides atpositions 11, 12, and 13 from the 5′ end of the antisense strand, and atleast two 2′-F nucleotides at positions 11, 12, and 13 from the 3′ endof the sense strand.

In some embodiments, one or more nucleotides of an Hif2α RNAi triggerare linked by non-standard linkages or backbones (i.e. modifiedinternucleoside linkages or modified backbones). In some embodiments, amodified internucleoside linkage is a non-phosphate-containing covalentinternucleoside linkage. Modified internucleoside linkages or backbonesinclude, but are not limited to, phosphorothioates, 5′-phosphorothioategroup (represented herein as a lower case ‘s’ before a nucleotide, as insN, sn, sNf, or sdN), chiral phosphorothioates, thiophosphate,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkyl-phosphonates,thionoalkylphosphotriesters, morpholino linkages, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein the adjacent pairs of nucleoside unitsare linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. In other embodiments, amodified internucleoside linkage or backbone lacks a phosphorus atom.Modified internucleoside linkages lacking a phosphorus atom include, butare not limited to, short chain alkyl or cycloalkyl inter-sugarlinkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages,or one or more short chain heteroatomic or heterocyclic inter-sugarlinkages. In some embodiments, modified internucleoside backbonesinclude, but are not limited to, siloxane backbones, sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones,methylene formacetyl and thioformacetyl backbones, alkene containingbackbones, sulfamate backbones, methyleneimino and methylenehydrazinobackbones, sulfonate and sulfonamide backbones, amide backbones; andothers having mixed N, O, S, and CH₂ component parts.

In some embodiments, an Hif2α RNAi trigger contains one or more modifiednucleotides and one or more modified internucleoside linkages. In someembodiments, a 2′-modified nucleotide is combined with modifiedinternucleoside linkage. For example, in some embodiments, a sensestrand of an Hif2α RNAi trigger can contain 1, 2, 3, 4 phosphorothioatelinkages, an antisense strand of a Hif2α RNAi trigger can contain 1, 2,3, or 4 phosphorothioate linkages, or both the sense strand and theantisense strand independently can contain 1, 2, 3, or 4phosphorothioate linkages.

In some embodiments, an Hif2α RNAi trigger sense strand contains twophosphorothioate internucleoside linkages. In some embodiments, the twophosphorothioate internucleoside linkages are between the nucleotides atpositions 1-3 from the 3′ end of the sense strand. In some embodiments,the two phosphorothioate internucleoside linkages are between thenucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5′ endof the sense strand. In some embodiments, an Hif2α RNAi triggerantisense strand contains four phosphorothioate internucleosidelinkages. In some embodiments, the four phosphorothioate internucleosidelinkages are between the nucleotides at positions 1-3 from the 5′ end ofthe sense strand and between the nucleotides at positions 19-21, 20-22,21-23, 22-24, 23-25, or 24-26 from the 5′ end. In some embodiments, anHif2α RNAi trigger contains two phosphorothioate internucleosidelinkages in the sense strand and four phosphorothioate internucleosidelinkages in the antisense strand.

In some embodiments, an Hif2α RNAi trigger is prepared or provided as asalt, mixed salt, or a free-acid.

Examples of antisense strands containing modified nucleotides areprovided in Table 2A and Table 5B. Examples of sense strands containingmodified nucleotides are provided in Table 2B and Table 5B. In Tables2A, 2B and 5B, the following notations are used to indicate modifiednucleotides:

-   -   N=2′-OH(unmodified)ribonucleotide (capital letter without or d        indication)    -   n=2′-OMe modified nucleotide    -   Nf=2′-fluoro modified nucleotide    -   A dN=2′-deoxynucleotides    -   N_(UNA)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)    -   NM=2′-methoxyethyl nucleotide    -   (invdN)=inverted deoxyribonucleotide (3′-3′ linked nucleotide)    -   (invAb)=inverted abasic nucleotide    -   s=phosphorothioate linked nucleotide    -   p=phosphate    -   vpdN=vinyl phosphonate deoxyribonucleotide

TABLE 2A Hif2α RNAi trigger antisense strands havingmodified nucleotides. Un- mod. SEQ  SEQ AntisenseAntisense Strand Sequence ID ID Strand ID (5′ → 3′) NO. NO. AM00159-ASdTGfgAfgCfuUfgUfgUfgUfuCfgCfad 112 21 TsdT AM00160-ASdTGfgUfaCfuGfgGfuGfgCfgUfaGfcd 113 22 TsdT AM00161-ASdTCfgUfuAfcGfuUfgAfcAfgGfuAfgd 106 17 TsdT AM00162-ASdTAfgCfuUfgUfgUfgUfuCfgCfaGfgd 103 13 TsdT AM00163-ASdTGfaGfcUfuGfuGfuGfuUfcGfcAfgd 110 19 TsdT AM00164-ASdTAfgGfaGfcUfuGfuGfuGfuUfcGfcd 104 14 TsdT AM00165-ASdTGfaGfgAfgCfuUfgUfgUfgUfuCfgd 111 20 TsdT AM00166-ASdTAfgAfgGfaGfcUfuGfuGfuGfuUfcd 102 12 TsdT AM00167-ASdTUfgUfcAfcGfaUfgCfgGfuGfgUfud 137 27 TsdT AM00168-ASdTAfcGfuUfgAfcAfgGfuAfgGfgUfud 101 11 TsdT AM00169-ASdTUfaCfgUfuGfaCfaGfgUfaGfgGfud 135 24 TsdT AM00170-ASdTUfcGfuUfaCfgUfuGfaCfaGfgUfad 136 25 TsdT AM00171-ASdTAfuCfgUfuAfcGfuUfgAfcAfgGfud 105 15 TsdT AM00172-ASdTAfaUfcGfuUfaCfgUfuGfaCfaGfgd 100 10 TsdT AM01770-ASdTGfuAfaAfaCfaAfuUfgUfgUfaCfud 114 23 TsdT AM01772-ASdTGfuAfaAUNAaCfaAfuUfgUfgUfaCf 116 23 udTsdT AM01773-ASdTGfuAfaAfAUNACfaAfuUfgUfgUfaC 115 23 fudTsdT AM01775-ASdTCfuAfgCfaAfcAfaAfaCfcUfuAfad 107 18 TsdT AM01777-ASdTCfuAfgCUNAaAfcAfaAfaCfcUfuAf 109 18 adTsdT AM01778-ASdTCfuAfgCfAUNAAfcAfaAfaCfcUfuA 108 18 fadTsdT AM01780-ASdTAfaAfuCfgUfuAfcGfuUfgAfcAfgd  94  7 TsdT AM01782-ASdTAfaAfuCUNAgUfuAfcGfuUfgAfcAf  96  7 sgdTdT AM01783-ASdTAfaAfuCfGUNAUfuAfcGfuUfgAfcA  95  7 fgdTsdT AM01784-ASdTUfuCfaUfgAfaAfuCfgUfuAfcGfud 138 30 TsdT AM01786-ASdTUfuCfaUUNAgAfaAfuCfgUfuAfcGf 145 30 udTsdT AM01787-ASdTUfuCfaUfGUNAAfaAfuCfgUfuAfcG 144 30 fudTsdT AM01789-ASdTAfaGfuUfaAfgCfuCfcCfaUfaCfad  97  9 TsdT AM01791-ASdTAfaGfuUUNAaAfgCfuCfcCfaUfaCf  99  9 adTsdT AM01792-ASdTAfaGfuUfAUNAAfgCfuCfcCfaUfaC  98  9 fadTsdT AM02090-ASdTUfuCfaUfgAUNAaAfuCfgUfuAfcGf 143 30 udTsdT AM02091-ASdTUfuCfaUfgAfAUNAAfuCfgUfuAfcG 142 30 fudTsdT AM02092-ASdTUfuCfaUfgAfaAfuCfgUfUUNAAfcG 139 30 fudTsdT AM02133-ASdTUfuCfaUfgAfaAfucgUfuAfcGfudT 141 30 sdT AM02140-ASdTsUfsuCfaUfgAfaAfucgUfuAfcGfu 128 31 Ufgsgsc AM02145-ASdTsUfsuCfaUfgAfaAfucgUfuAfcGfu 127 33 UfggscsuGu AM02146-ASdTsUfsuCfaUfgAfaAfucgUfuAfcGfu 123 29 CfggscsuGu AM02147-ASdTsUfsuCfaUfgAfaAfucgUfuAfcGfu 126 32 UfggscsuAu AM02150-ASdTsUfsuCfaUfgAfaAfucgUfuAfcGfu 125 34 UfggcusdTsdT AM02327-ASpdTUfuCfaUfgAfaAfuCfgUfuAfcGfu 147 30 dTsdT AM02341-ASdTsUfsuCfaUfgAUNAaAfuCfgUfuAfc 130 28 GfuCfggscsuAu AM02342-ASdTsUfuCfaUfgAUNAaAfuCfgUfuAfcG 134 28 fuCfggcsuAu AM02345-ASdTsUfsuCfaUfgAfAUNAAfuCfgUfuAf 129 28 cGfuCfggscsuAu AM02346-ASdTsUfuCfaUfgAfAUNAAfuCfgUfuAfc 133 28 GfuCfggcsuAu AM02508-ASdTsUfsuCfaUfgAfaAfuCfgUfuAfcGf 120 30 udTsdT AM02509-ASdTUfuCfaUfgAfaAfucGfUfuAfcGfud 140 30 TsdT AM02523-ASdTsAfsaCfcAfcAfuAfcGfuUfgGfaGf 117  8 udTsdT AM02525-ASdTsUfsgAfuAfaAfcAfcUfuAfaCfcCf 119 26 adTsdT AM02527-ASdTsCfsaUfgAfaAfuCfgUfuAfcGfuUf 118 16 gdTsdT AM02529-ASusCfsaUfgAfaAfuCfgUfuAfcGfuUfg 148 35 dTsdT AM02604-ASdTsUfsuCfaUfgAfaAfucGfUfuAfcGf 122 30 udTsdT AM02605-ASdTsUfsuCfaUfgAfaAfucgUfuAfcGfu 124 30 dTsdT AM02848-ASdTsUfuCfaUfgAfaAfucGfUfuAfcGfu 132 30 dTsdT AM02849-ASdTsUfuCfaUfgAfaAfuCfgUfUfacGfu 131 30 dTsdT AM02850-ASdTsUfsuCfaUfgAfaAfuCfgUfUfacGf 121 30 udTsdT AM02998-ASusUfsuCfaUfgAfaAfucgUfuAfcGfud 160 40 TsdT AM03000-ASusCfsuAfgCfaAfcAfaaAfCfcUfuAfa 151 36 dTsdT AM03001-ASusCfsuAfgCfaaCfAfaaAfCfcUfuAfa 149 36 dTsdT AM03002-ASusCfsuAfgCfaAfcAfaAfaCfCfuuAfa 152 36 dTsdT AM03003-ASusCfsuAfgCfaaCfAfaAfaCfCfuuAfa 150 36 dTsdT AM03008-ASusGfsuAfaAfaCfaAfuuGfUfgUfaCfu 154 37 dTsdT AM03009-ASusGfsuAfaAfaCfaAfuUfgUfGfuaCfu 153 37 dTsdT AM03059-ASpdTsUfsuCfaUfgAfaAfucgUfuAfcGf 146 30 udTsdT AM03465-ASusUfsuCfaUfgAfaAfucgUfuAfcGfus 161 41 usg AM03513-ASusUfsuCfaUfgAfaAfucguuacgususg 162 41 AM03514-ASusUfsuCfaUfgAfaaucguuacgususg 165 41 AM03517-ASasUfsuCfaUfgAfaAfucgUfuAfcGfus  90  4 usg AM03685-ASusUfsuCfaUfgAfaAfucgUfuacgusus 159 41 g AM03688-ASusUfsUfCfaugAfaAfucgUfuacgusus 167 41 g AM03689-ASusUfsuCfaUfgAfaaucgUfuacgususg 163 41 AM03690-ASusUfsUfcaugAfaAfucgUfuacgususg 166 41 AM04001-ASusGfsuAfaAfaCfaAfuugUfgUfaCfus 155 38 usu AM04004-ASasGfsuAfaAfaCfaAfuugUfgUfaCfus  84  1 usu AM04007-ASusGfsuAfaAfaCfaAfuugUfgUfaCfuu 156 39 usasa AM04010-ASasGfsuAfaAfaCfaAfuugUfgUfaCfuu  85  2 usasa AM04015-ASasGfsuAfaAfaCfaAfuugUfguaCfuuu  83  2 sasa AM04018-ASasGfsuAfaAfaCfaauugUfguacuuusa  86  2 sa AM04040-ASvpusUfsuCfaUfgAfaAfucgUfuAfcGf 168 41 ususg AM04101-ASasUfsuCfaUfgAfaaucgUfuacgususg  91  4 AM04102-ASusUfsuCfaUfgAfaaucgUfuacguugsa 164 42 s(invdT) AM04103-ASasUfsuCfaUfgAfaaucgUfuacguugsa  92  5 s(invdT) AM04104-ASusUfsucaugAfaAfucgUfuacgususg 157 41 AM04105-ASasUfsucaugAfaAfucgUfuacgususg  88  4 AM04106-ASasUfsucaugAfaAfucgUfuacgusTMsG  87  3 M AM04244-ASusUfsuCfaUfgaAfaucgUfuacgususg 158 41 AM04452-ASasUfsuCfaUfgAfaaucgUfuAfcguugs  93  6 gsc AM04455-ASasUfsuCfaUfgaAfaucgUfuAfcguugs  89  6 gsc

TABLE 2B Hif2α RNAi trigger sense strands having modified nucleotides.Unmod. Sense SEQ SEQ Strand ID ID ID SS Sequence 5′ → 3′ NO. NO.AM00158-SS CfuGfuCfaAfcGfuAfaCfgAfuUfuAf(invdT) 277 67 AM00188-SS(NH2-C6)uAuUfgCfgAfaCfaCfaCfaAfgCfuCfcAf(invdT) 265 79 AM00189-SS(NH2-C6)uAuGfcUfaCfgCfcAfcCfcAfgUfaCfcAf(invdT) 264 70 AM00190-SS(NH2-C6)uAuCfuAfcCfuGfuCfaAfcGfuAfaCfgAf(invdT) 263 65 AM00191-SS(NH2-C6)uAuCfcUfgCfgAfaCfaCfaCfaAfgCfuAf(invdT) 262 62 AM00207-SS(NH2-C6)CfuGfcGfaAfcAfcAfcAfaGfcUfcAf(invdT) 258 66 AM00208-SS(NH2-C6)GfcGfaAfcAfcAfcAfaGfcUfcCfuAf(invdT) 260 69 AM00209-SS(NH2-C6)CfgAfaCfaCfaCfaAfgCfuCfcUfcAf(invdT) 256 64 AM00210-SS(NH2-C6)GfaAfcAfcAfcAfaGfcUfcCfuCfuAf(invdT) 259 68 AM00211-SS(NH2-C6)CfaGfuGfcAfaCfgCfcAfcCfcAfgAf(invdT) 253 61 AM00212-SS(NH2-C6)AfaCfcCfuAfcCfuGfuCfaAfcGfuAf(invdT) 247 45 AM00213-SS(NH2-C6)AfcCfcUfaCfcUfgUfcAfaGfgUfaAf(invdT) 248 48 AM00214-SS(NH2-C6)UfcAfcUfgUfcAfaCfgUfaAfcGfaAf(invdT) 266 78 AM00219-SS(NH2-C6)AfcCfuGfuCfaAfcGfuAfaCfgUfaAf(invdT) 249 49 AM00220-SS(NH2-C6)CfcUfgUfcAfaCfgUfaAfcGfaUfuAf(invdT) 255 63 AM00221-SS(NH2-C6)UfgCfgAfaCfaCfaCfaAfgCfuCfcAf(invdT) 267 79 AM00222-SS(NH2-C6)GfcUfaCfgCfcAfcCfcAfgUfaCfcAf(invdT) 261 70 AM00223-SS(NH2-C6)CfuAfcCfuGfuCfaAfcGfuAfaCfgAf(invdT) 257 65 AM00224-SS(NH2-C6)CfcUfgCfgAfaCfaCfaCfaAfgCfuAf(invdT) 254 62 AM00366-SS(Alk-SS-C6)CfuGfcGfaAfcAfcAfcAfaGfcUfcAf(invdT) 220 66 AM00367-SS(Alk-SS-C6)GfcGfaAfcAfcAfcAfaGfcUfcCfuAf(invdT) 223 69 AM00369-SS(Alk-SS-C6)GfaAfcAfcAfcAfaGfcUfcCfuCfuAf(invdT) 222 68 AM00530-SS(Alk-SS-C6)AfcCfcUfaCfcUfgUfcAfaGfgUfaAf(invdT) 212 48 AM00531-SS(Alk-SS-C6)UfcAfcUfgUfcAfaCfgUfaAfcGfaAf(invdT) 224 78 AM00543-SS(Alk-SS-C6)AfaCfcCfuAfcCfuGfuCfaAfcGfuAf(invdT) 211 45 AM00544-SS(Alk-SS-C6)AfcCfuGfuCfaAfcGfuAfaCfgUfaAf(invdT) 213 49 AM00545-SS(Alk-SS-C6)CfuAfcCfuGfuCfaAfcGfuAfaCfgAf(invdT) 219 65 AM01771-SSAfgUfaCfaCfaAfuUfgUfuUfuAfcAf(invdT) 271 56 AM01776-SSUfuAfaGfgUfuUfuGfuUfgCfuAfgAf(invdT) 284 82 AM01785-SSAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT) 269 51 AM01790-SSUfgUfaUfgGfgAfgCfuUfaAfcUfuAf(invdT) 283 81 AM01859-SS(Alk-SS-C6)AfgUfaCfaCfaAfuUfgUfuUfuAfcAf(invdT) 218 56 AM01860-SS(Alk-SS-C6)UfuAfaGfgUfuUfuGfuUfgCfuAfgAf(invdT) 226 82 AM01861-SS(Alk-SS-C6)CfuGfuCfaAfcGfuAfaCfgAfuUfuAf(invdT) 221 67 AM01862-SS(Alk-SS-C6)AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT) 215 51 AM01863-SS(Alk-SS-C6)UfgUfaUfgGfgAfgCfuUfaAfcUfuAf(invdT) 225 81 AM01994-SS(Alk-C6)uAuAfcCfuGfuCfaAfcGfuAfaCfgUfaAf(invdT) 169 71 AM02043-SS(Me-Alk-SS-C6)AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT) 231 51 AM02093-SS(DBCO-TEG)uAuAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT) 228 72 AM02135-SS(Alk-SS-C6)AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT) 216 51 AM02137-SS(Alk-SS-C6)AfcGfuAfAfCfgAfuUfuCfaUfgAfaAf(invdT) 217 51 AM02139-SS(Alk-SS-C6)AfcGfuaaCfgAfuUfuCfaUfgAfaAf(invdT) 214 51 AM02142-SSCfsasAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 276 59 AM02144-SSCUNAsasAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 278 59 AM02149-SSuAuAusCfsaAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 280 75 AM02163-SSuAuAusCfsgAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 282 76 AM02363-SS(DBCO-TEG)uAuAusCfsgAfcGfuAfaCfgAfuUfuCfaUfgAfa(invdA) 230 76 AM02364-SSuAuAusCfsgAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 281 76 AM02365-SSuAuAusCfgAfcGfuAfaCfgAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 279 76 AM02456-SS(Alk-SMPT-C6)AfcGfuAfaCfgAfuUfuCfaUfgAfaAf(invdT) 193 51 AM02510-SS(Me-Alk-SS-C6)AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT) 232 51 AM02512-SS(Me-Alk-SS-C6)AfcGfuAfacGfAfuUfuCfaUfgAfaAf(invdT) 233 51 AM02522-SS(NH2-C6)AfcUfcCfaAfcGfuAfuGfuGfgUfuAf(invdT) 250 55 AM02524-SS(NH2-C6)UfgGfgUfuAfaGfuGfuUfuAfuCfaAf(invdT) 268 80 AM02526-SS(NH2-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfgAf(invdT) 252 60 AM02528-SS(NH2-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfg(invdA) 251 58 AM02546-SS(Me-Alk-SS-C6)AfcUfcCfaAfcGfuAfuGfuGfgUfuAf(invdT) 235 55 AM02547-SS(Me-Alk-SS-C6)UfgGfgUfuAfaGfuGfuUfuAfuCfaAf(invdT) 242 80 AM02548-SS(Me-Alk-SS-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfgAf(invdT) 241 60 AM02549-SS(Me-Alk-SS-C6)CfaAfcGfuAfaCfgAfuUfuCfaUfg(invdA) 240 58 AM02852-SSAfscGfuAfacGfAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 272 50 AM02853-SSAfscGfUfaaCfgAfuUfuCfaUfgAfaAf(C6-SS-Alk-Me) 273 50 AM02856-SS(Me-Alk-SS-C6)AfcGfUfaaCfgAfuUfuCfaUfgAfaAf(invdT) 234 51 AM02999-SS(DBCO-TEG)uAuAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT) 229 72 AM03004-SS(Me-Alk-SS-C6)UfuAfaGfguUfUfuGfuUfgCfuAfgAf(invdT) 244 82 AM03005-SS(Me-Alk-SS-C6)uuAfaGfguUfUfuguUfgCfuAfga(invdT) 246 82 AM03006-SS(Me-Alk-SS-C6)UfuAfAfggUfuUfuGfuUfgCfuAfgAf(invdT) 243 82 AM03007-SS(Me-Alk-SS-C6)uuAfAfggUfuUfuguUfgCfuAfga(invdT) 245 82 AM03010-SS(Me-Alk-SS-C6)AfgUfaCfacAfAfuUfgUfuUfuAfcAf(invdT) 236 56 AM03011-SS(Me-Alk-SS-C6)agUfaCfacAfAfuUfgUfuuuAfca(invdT) 238 56 AM03012-SS(Me-Alk-SS-C6)AfgUfAfcaCfaAfuUfgUfuUfuAfcAf(invdT) 237 56 AM03013-SS(Me-Alk-SS-C6)agUfAfcaCfaAfuUfgUfuuuAfca(invdT) 239 56 AM03058-SSAfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT) 270 51 AM03307-SSagUfAfcaCfaAfuUfgUfuuuAfca(invdT) 274 56 AM03308-SS(Alk-C6-C6)AfcGfuAfaCfGfAfuUfuCfaUfgAfaAf(invdT) 170 51 AM03467-SS(Alk-SMPT-C6)AfscGfuAfaCfGfAfuUfuCfaUfgAfaa(invdT) 194 51 AM03469-SS(Alk-SMPT-C6)ascGfuAfaCfGfAfuuuCfaUfgAfaa(invdT) 201 51 AM03471-SS(Alk-SMPT-C6)ascguAfaCfgAfuuuCfaUfgAfaa(invdT) 208 51 AM03473-SS(Alk-SMPT-C6)ascGfuaaCfgAfuuuCfaugAfaa(invdT) 200 51 AM03475-SS(Alk-SMPT-C6)ascguaaCfgAfuuuCfaugaaa(invdT) 204 51 AM03516-SS(Alk-SMPT-C6)ascguaaCfgAfuuucaugaaa(invdT) 203 51 AM03519-SS(Alk-SMPT-C6)ascGfuAfaCfGfAfuuuCfaUfgAfau(invdT) 202 54 AM03687-SS(Alk-SMPT-C6)ascguaaCfGfAfuuucaugaaa(invdT) 205 51 AM03692-SS(Alk-PEGS-C6)uAuascguaaCfGfAfuuucaugaaa(invdT) 185 72 AM03694-SS(Alk-PEGS-C6)uAuascGfuAfaCfGfAfuuuCfaUfgAfau(invdT) 183 73 AM03708-SS(Alk-PEGS-C6)uAuascGfuAfaCfGfAfuuuCfaUfgAfaa(invdT) 182 72 AM03710-SS(Alk-PEGS-C6)uAuascguaaCfGfAfuuuCfaugaaa(invdT) 189 72 AM03712-SS(Alk-PEGS-C6)uAuascguaaCfGfAfuuuCfaugaau(invdT) 190 73 AM03714-SS(Alk-PEG5-C6)(Alk-PEG5-Ser)- 180 72 uAuascguaaCfGfAfuuuCfaugaaa(invdT)AM03774-SS (Alk-C6-SMPT-C6)ascguaaCfgAfuuuCfaugaaa(invdT) 174 51AM03829-SS (Alk-PEGS-C6)uAuascguaaCfgAfuuuCfaugaaa(invdT) 184 72AM03830-SS (Chol-TEG)uAuascguaaCfgAfuuuCfaugaaa(invdT) 227 72 AM03831-SSascguaaCfgAfuuuCfaugaaa(NAG13) 275 50 AM04003-SS(Alk-SMPT-C6)asgUfaCfaCfAfAfuUfgUfuuuAfca(invdT) 209 56 AM04006-SS(Alk-SMPT-C6)asgUfaCfaCfAfAfuUfgUfuuuAfcu(invdT) 210 57 AM04009-SS(Alk-SMPT-C6)asaAfgUfaCfaCfAfAfuUfgUfuuuAfc(invdA) 195 43 AM04012-SS(Alk-SMPT-C6)asaAfgUfaCfaCfAfAfuUfgUfuuuAfc(invdT) 196 44 AM04014-SS(Alk-SMPT-C6)asaaguaCfaCfAfAfuuguuuuac(invdT) 198 44 AM04017-SS(Alk-SMPT-C6)asaagUfaCfaCfAfAfuUfgUfuuuac(invdT) 199 44 AM04020-SS(Alk-SMPT-C6)asaaguacaCfAfAfuuguuuuac(invdT) 197 44 AM04107-SS(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaaa(invdT) 175 51 AM04107-SS(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaaa(invdT) 175 51 AM04109-SS(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaAMTM(invdT) 176 52 AM04111-SS(Alk-C6-SMPT-C6)AMsCMguaaCfGfAfuuucaugaAMTM(invdT) 171 52 AM04113-SS(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaasus(invdT) 178 54 AM04115-SS(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaasus(invAb) 177 53 AM04117-SS(Alk-C6-SMPT-C6)ascguaaCfGfAfuuucaugaau(invdT) 179 54 AM04119-SS(Alk-C6-SMPT-C6)asacguaaCfGfAfuuucaugaau(invdT) 173 47 AM04121-SS(Alk-C6-SMPT-C6)asacguaaCfGfAfuuucaugaaa(invdT) 172 46 AM04122-SS(Alk-PEGS-C6)ascguaaCfGfAfuuucaugaaa(invdT) 181 51 AM04241-SS(Alk-SMPT-C6)ascguaaCfGfAfuuucaugaaAM(invdT) 206 51 AM04243-SS(Alk-SMPT-C6)ascguaaCfGfAfuuucaugaaAMs(invdT) 207 51 AM04246-SS(Alk-PEG5-C6)uAuascguaaCfGfAfuuucaugaaAMs(invdT) 186 72 AM04248-SS(Alk-PEG5-C6)uauascguaaCfGfAfuuucaugaaAMs(invdT) 187 72 AM04451-SS(Alk-PEG5-C6)uAuascguaaCfGfAfuuucaugaaudTs(invdT) 188 74 AM04454-SS(Alk-PEG5-C6)uAucsasacguAfaCfGfAfuuucaugAfaudTs(invdT) 192 77 AM04457-SS(Alk-PEG5-C6)uAucsaacguAfaCfGfAfuuucaugAfaudTs(invdT) 191 77 AM03710-SS(Alk-PEG5-C6)uAuascguaaCfGfAfuuuCfaugaaa(invdT) 189 72

TABLE 3 Hif2α RNAi trigger duplexes with Duplex ID numbers. SEQ Unmod.SEQ Unmod. Duplex Antisense ID SEQ Sense Strand ID SEQ ID Strand ID NO.ID NO. ID NO. ID NO. AD00086 AM00159-AS 112 21 AM00188-SS 265 79 AD00087AM00160-AS 113 22 AM00189-SS 264 70 AD00088 AM00161-AS 106 17 AM00190-SS263 65 AD00089 AM00162-AS 103 13 AM00191-SS 262 62 AD00102 AM00163-AS110 19 AM00207-SS 258 66 AD00103 AM00164-AS 104 14 AM00208-SS 260 69AD00104 AM00165-AS 111 20 AM00209-SS 256 64 AD00105 AM00166-AS 102 12AM00210-SS 259 68 AD00106 AM00167-AS 137 27 AM00211-SS 253 61 AD00107AM00168-AS 101 11 AM00212-SS 247 45 AD00108 AM00169-AS 135 24 AM00213-SS248 48 AD00109 AM00170-AS 136 25 AM00214-SS 266 78 AD00110 AM00171-AS105 15 AM00219-SS 249 49 AD00111 AM00172-AS 100 10 AM00220-SS 255 63AD00112 AM00159-AS 112 21 AM00221-SS 267 79 AD00113 AM00160-AS 113 22AM00222-SS 261 70 AD00114 AM00161-AS 106 17 AM00223-SS 257 65 AD00115AM00162-AS 103 13 AM00224-SS 254 62 AD00215 AM00163-AS 110 19 AM00369-SS222 68 AD00268 AM00166-AS 102 12 AM00369-SS 222 68 AD00269 AM00169-AS135 24 AM00530-SS 212 48 AD00270 AM00170-AS 136 25 AM00531-SS 224 78AD00274 AM00168-AS 101 11 AM00543-SS 211 45 AD00275 AM00171-AS 105 15AM00544-SS 213 49 AD00276 AM00161-AS 106 17 AM00545-SS 219 65 AD00285AM00163-AS 110 19 AM00366-SS 220 66 AD00286 AM00164-AS 104 14 AM00367-SS223 69 AD00373 AM00169-AS 135 24 AM00659-SS 212 48 AD00374 AM00170-AS136 25 AM00660-SS 224 78 AD00375 AM00168-AS 101 11 AM00679-SS 211 45AD00376 AM00171-AS 105 15 AM00661-SS 213 49 AD00377 AM00161-AS 106 17AM00662-SS 219 65 AD00988 AM01772-AS 116 23 AM01771-SS 271 56 AD00989AM01777-AS 109 18 AM01776-SS 284 82 AD00990 AM01782-AS 96 7 AM00158-SS277 67 AD00991 AM01786-AS 145 30 AM01785-SS 269 51 AD00992 AM01791-AS 999 AM01790-SS 283 81 AD00993 AM01773-AS 115 23 AM01771-SS 271 56 AD00994AM01778-AS 108 18 AM01776-SS 284 82 AD00995 AM01783-AS 95 7 AM00158-SS277 67 AD00996 AM01787-AS 144 30 AM01785-SS 269 51 AD00997 AM01792-AS 989 AM01790-SS 283 81 AD01020 AM01772-AS 116 23 AM01859-SS 218 56 AD01021AM01773-AS 115 23 AM01859-SS 218 56 AD01022 AM01770-AS 114 23 AM01859-SS218 56 AD01023 AM01777-AS 109 18 AM01860-SS 226 82 AD01024 AM01778-AS108 18 AM01860-SS 226 82 AD01025 AM01775-AS 107 18 AM01860-SS 226 82AD01026 AM01782-AS 96 7 AM01861-SS 221 67 AD01027 AM01783-AS 95 7AM01861-SS 221 67 AD01028 AM01780-AS 94 7 AM01861-SS 221 67 AD01029AM01786-AS 145 30 AM01862-SS 215 51 AD01030 AM01787-AS 144 30 AM01862-SS215 51 AD01031 AM01784-AS 138 30 AM01862-SS 215 51 AD01032 AM01791-AS 999 AM01863-SS 225 81 AD01033 AM01792-AS 98 9 AM01863-SS 225 81 AD01034AM01789-AS 97 9 AM01863-SS 225 81 AD01180 AM00171-AS 105 15 AM01994-SS169 71 AD01214 AM01784-AS 138 30 AM02043-SS 231 51 AD01255 AM01784-AS138 30 AM02093-SS 228 72 AD01256 AM02090-AS 143 30 AM01862-SS 215 51AD01257 AM02091-AS 142 30 AM01862-SS 215 51 AD01258 AM02092-AS 139 30AM01862-SS 215 51 AD01288 AM02133-AS 141 30 AM02135-SS 216 51 AD01289AM01784-AS 138 30 AM02137-SS 217 51 AD01290 AM01784-AS 138 30 AM02139-SS214 51 AD01291 AM02140-AS 128 31 AM02142-SS 276 59 AD01292 AM02140-AS128 31 AM02144-SS 278 59 AD01293 AM02145-AS 127 33 AM02149-SS 280 75AD01294 AM02146-AS 123 29 AM02163-SS 282 76 AD01295 AM02147-AS 126 32AM02149-SS 280 75 AD01296 AM02150-AS 125 34 AM02149-SS 280 75 AD01391AM01784-AS 138 30 AM01785-SS 269 51 AD01392 AM02327-AS 147 30 AM01785-SS269 51 AD01404 AM02341-AS 130 28 AM02364-SS 281 76 AD01405 AM02341-AS130 28 AM02365-SS 279 76 AD01406 AM02342-AS 134 28 AM02364-SS 281 76AD01407 AM02342-AS 134 28 AM02365-SS 279 76 AD01408 AM02345-AS 129 28AM02364-SS 281 76 AD01409 AM02345-AS 129 28 AM02365-SS 279 76 AD01410AM02346-AS 133 28 AM02364-SS 281 76 AD01411 AM02346-AS 133 28 AM02365-SS279 76 AD01424 AM02345-AS 129 28 AM02363-SS 230 76 AD01476 AM01784-AS138 30 AM02456-SS 193 51 AD01522 AM02133-AS 141 30 AM02510-SS 232 51AD01523 AM02509-AS 140 30 AM02512-SS 233 51 AD01524 AM02508-AS 120 30AM02043-SS 231 51 AD01525 AM02523-AS 117 8 AM02522-SS 250 55 AD01526AM02525-AS 119 26 AM02524-SS 268 80 AD01527 AM02527-AS 118 16 AM02526-SS252 60 AD01528 AM02529-AS 148 35 AM02528-SS 251 58 AD01546 AM02523-AS117 8 AM02546-SS 235 55 AD01547 AM02525-AS 119 26 AM02547-SS 242 80AD01548 AM02527-AS 118 16 AM02548-SS 241 60 AD01549 AM02529-AS 148 35AM02549-SS 240 58 AD01554 AM02604-AS 122 30 AM02512-SS 233 51 AD01555AM02605-AS 124 30 AM02510-SS 232 51 AD01654 AM02848-AS 132 30 AM02852-SS272 50 AD01655 AM02848-AS 132 30 AM02512-SS 233 51 AD01656 AM02849-AS131 30 AM02853-SS 273 50 AD01657 AM02849-AS 131 30 AM02856-SS 234 51AD01658 AM02850-AS 121 30 AM02853-SS 273 50 AD01659 AM02850-AS 121 30AM02856-SS 234 51 AD01884 AM02998-AS 160 40 AM02510-SS 232 51 AD01885AM02998-AS 160 40 AM02999-SS 229 72 AD01886 AM03000-AS 151 36 AM03004-SS244 82 AD01887 AM03001-AS 149 36 AM03005-SS 246 82 AD01888 AM03002-AS152 36 AM03006-SS 243 82 AD01889 AM03003-AS 150 36 AM03007-SS 245 82AD01890 AM03008-AS 154 37 AM03010-SS 236 56 AD01891 AM03008-AS 154 37AM03011-SS 238 56 AD01892 AM03009-AS 153 37 AM03012-SS 237 56 AD01893AM03009-AS 153 37 AM03013-SS 239 56 AD01910 AM02605-AS 124 30 AM03058-SS270 51 AD01911 AM03059-AS 146 30 AM03058-SS 270 51 AD02073 AM03009-AS153 37 AM03307-SS 274 56 AD02074 AM02605-AS 124 30 AM03308-SS 170 51AD02691 AM03465-AS 161 41 AM03467-SS 194 51 AD02692 AM03465-AS 161 41AM03469-SS 201 51 AD02693 AM03465-AS 161 41 AM03471-SS 208 51 AD02694AM03465-AS 161 41 AM03473-SS 200 51 AD02695 AM03465-AS 161 41 AM03475-SS204 51 AD02733 AM03513-AS 162 41 AM03516-SS 203 51 AD02734 AM03514-AS165 41 AM03516-SS 203 51 AD02735 AM03517-AS 90 4 AM03519-SS 202 54AD02857 AM03685-AS 159 41 AM03687-SS 205 51 AD02858 AM03688-AS 167 41AM03687-SS 205 51 AD02859 AM03689-AS 163 41 AM03687-SS 205 51 AD02860AM03690-AS 166 41 AM03687-SS 205 51 AD02861 AM03685-AS 159 41 AM03692-SS185 72 AD02862 AM03517-AS 90 4 AM03694-SS 183 73 AD02873 AM03465-AS 16141 AM03708-SS 182 72 AD02874 AM03465-AS 161 41 AM03710-SS 189 72 AD02875AM03517-AS 90 4 AM03712-SS 190 73 AD02876 AM03465-AS 161 41 AM03714-SS180 72 AD02949 AM03465-AS 161 41 AM03774-SS 174 51 AD03011 AM03465-AS161 41 AM03829-SS 184 72 AD03012 AM03465-AS 161 41 AM03830-SS 227 72AD03013 AM03465-AS 161 41 AM03831-SS 275 50 AD03187 AM04001-AS 155 38AM04003-SS 209 56 AD03188 AM04004-AS 84 1 AM04006-SS 210 57 AD03189AM04007-AS 156 39 AM04009-SS 195 43 AD03190 AM04010-AS 85 2 AM04012-SS196 44 AD03191 AM04010-AS 85 2 AM04014-SS 198 44 AD03192 AM04015-AS 83 2AM04017-SS 199 44 AD03193 AM04018-AS 86 2 AM04020-SS 197 44 AD03215AM04040-AS 168 41 AM03469-SS 201 51 AD03216 AM04040-AS 168 41 AM03475-SS204 51 AD03253 AM03689-AS 163 41 AM04107-SS 175 51 AD03254 AM04101-AS 914 AM04117-SS 179 54 AD03255 AM04102-AS 164 42 AM04121-SS 172 46 AD03256AM04103-AS 92 5 AM04119-SS 173 47 AD03257 AM04104-AS 157 41 AM04107-SS175 51 AD03258 AM04105-AS 88 4 AM04117-SS 179 54 AD03259 AM04106-AS 87 3AM04109-SS 176 52 AD03260 AM04105-AS 88 4 AM04109-SS 176 52 AD03261AM04105-AS 88 4 AM04111-SS 171 52 AD03262 AM04105-AS 88 4 AM04113-SS 17854 AD03263 AM04105-AS 88 4 AM04115-SS 177 53 AD03264 AM03690-AS 166 41AM04107-SS 175 51 AD03265 AM03689-AS 163 41 AM03692-SS 185 72 AD03266AM03689-AS 163 41 AM04122-SS 181 51 AD03345 AM03689-AS 163 41 AM04241-SS206 51 AD03346 AM03689-AS 163 41 AM04243-SS 207 51 AD03347 AM04244-AS158 41 AM04243-SS 207 51 AD03348 AM03689-AS 163 41 AM04246-SS 186 72AD03349 AM03689-AS 163 41 AM04248-SS 187 72 AD03505 AM04101-AS 91 4AM04451-SS 188 74 AD03506 AM04452-AS 93 6 AM04454-SS 192 77 AD03507AM04455-AS 89 6 AM04454-SS 192 77 AD03508 AM04452-AS 93 6 AM04457-SS 19177

A sense strand containing a sequence listed in Table 2B can behybridized to any antisense strand containing a sequence listed in Table2A provided the two sequences have a region of at least 90%complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotidesequence. Representative Hif2α RNA triggers are represented by theDuplex ID Nos. shown in Table 3. In some embodiments an Hif2α RNAitrigger consists of any of the Duplex ID Nos. presented herein. In someembodiments an Hif2α RNAi trigger comprises of any of the Duplex ID Nos.presented herein. In some embodiments, an Hif2α RNAi trigger comprisesthe sense strand and antisense strand nucleotide sequences of any of theDuplex ID Nos. presented herein. In some embodiments, an Hif2α RNAitrigger comprises the sense strand and antisense strand nucleotidesequences of any of the Duplex ID Nos. presented herein and a targetinggroup and/or linking group wherein the targeting group and/or linkinggroup is covalently linked to the sense strand or the antisense strand.In some embodiments, an Hif2α RNAi trigger comprises the sense strandand antisense strand modified nucleotide sequences of any of the DuplexID Nos. presented herein. In some embodiments, an Hif2α RNAi triggercomprises the sense strand and antisense strand modified nucleotidesequences of any of the Duplex ID Nos. presented herein and a targetinggroup and/or linking group wherein the targeting group and/or linkinggroup is covalently linked to the sense strand or the antisense strand.

In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO.4. In some embodiments, a Hif2α RNAi trigger comprises an antisensestrand comprising a nucleotide base sequence of nucleotides 2-21 of SEQID NO. 4 and a sense strand comprising a nucleotide base sequence of SEQID NO. 53.

In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising a nucleotide base sequence of SEQ ID NO. 88, SEQ ID NO. 157,SEQ ID NO. 159, or SEQ ID NO. 163. In some embodiments, a Hif2α RNAitrigger comprises an antisense strand comprising a nucleotide basesequence of SEQ ID NO. 88 and a sense strand comprising a nucleotidebase sequence of SEQ ID NO. 179. In some embodiments, a Hif2α RNAitrigger comprises an antisense strand comprising a nucleotide basesequence of SEQ ID NO. 88 and a sense strand comprising a nucleotidebase sequence of SEQ ID NO. 177. In some embodiments, a Hif2α RNAitrigger comprises an antisense strand comprising a nucleotide basesequence of SEQ ID NO. 157 and a sense strand comprising a nucleotidebase sequence of SEQ ID NO. 175. In some embodiments, a Hif2α RNAitrigger comprises an antisense strand comprising a nucleotide basesequence of SEQ ID NO. 159 and a sense strand comprising a nucleotidebase sequence of SEQ ID NO. 185. In some embodiments, a Hif2α RNAitrigger comprises an antisense strand comprising a nucleotide basesequence of SEQ ID NO. 163 and a sense strand comprising a nucleotidebase sequence of SEQ ID NO. 185.

In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising SEQ ID NO. 88, SEQ ID NO. 157, SEQ ID NO. 159, or SEQ ID NO.163. In some embodiments, a Hif2α RNAi trigger comprises an antisensestrand comprising SEQ ID NO. 88 and a sense strand comprising SEQ ID NO.179. In some embodiments, a Hif2α RNAi trigger comprises an antisensestrand comprising SEQ ID NO. 88 and a sense strand comprising SEQ ID NO.177. In some embodiments, a Hif2α RNAi trigger comprises an antisensestrand comprising SEQ ID NO. 157 and a sense strand comprising SEQ IDNO. 175. In some embodiments, a Hif2α RNAi trigger comprises anantisense strand comprising SEQ ID NO. 159 and a sense strand comprisingSEQ ID NO. 185. In some embodiments, a Hif2α RNAi trigger comprises anantisense strand comprising SEQ ID NO. 163 and a sense strand comprisingSEQ ID NO. 185.

In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising a nucleotide base sequence of nucleotides 2-21 of SEQ ID NO.38. In some embodiments, a Hif2α RNAi trigger comprises an antisensestrand comprising a nucleotide base sequence of nucleotides 2-21 of SEQID NO. 38 and a sense strand comprising a nucleotide base sequence ofnucleotides 1-19 of SEQ ID NO. 56.

In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising a nucleotide base sequence of SEQ ID NO. 86, SEQ ID NO. 155,SEQ ID NO. 156. In some embodiments, a Hif2α RNAi trigger comprises anantisense strand comprising a nucleotide base sequence of SEQ ID NO. 156and a sense strand comprising a nucleotide base sequence of SEQ ID NO.195. In some embodiments, a Hif2α RNAi trigger comprises an antisensestrand comprising a nucleotide base sequence of SEQ ID NO. 86 and asense strand comprising a nucleotide base sequence of SEQ ID NO. 197. Insome embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising a nucleotide base sequence of SEQ ID NO. 155 and a sensestrand comprising a nucleotide base sequence of SEQ ID NO. 209.

In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising SEQ ID NO. 86, SEQ ID NO. 155, SEQ ID NO. 156. In someembodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising SEQ ID NO. 156 and a sense strand comprising SEQ ID NO. 195.In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising SEQ ID NO. 86 and a sense strand comprising SEQ ID NO. 197.In some embodiments, a Hif2α RNAi trigger comprises an antisense strandcomprising SEQ ID NO. 155 and a sense strand comprising SEQ ID NO. 209.

In some embodiments, an Hif2α RNAi trigger contains or is conjugated toa targeting group, linking group, delivery polymer, delivery vehicle,and/or other non-nucleotide group. The targeting group, linking group,delivery polymer, delivery vehicle, and/or other non-nucleotide groupcan be covalently linked to the 3′ and/or 5′ end of either the sensestrand and/or the antisense strand. In some embodiments, an Hif2α RNAitrigger can contains a targeting group, linking group, delivery polymer,delivery vehicle, or other non-nucleotide group linked to the 3′ and/or5′ end of the sense strand. In some embodiments a targeting group,linking group, delivery polymer, delivery vehicle, or othernon-nucleotide group is linked to the 5′ end of an Hif2α RNAi triggersense strand. In some embodiments, the targeting group, linking group,delivery polymer, delivery vehicle, and/or other non-nucleotide group islinked directly or indirectly to the trigger via a linker/linking group.In some embodiments, targeting group, linking group, delivery polymer,delivery vehicle, and/or other non-nucleotide group is linked to thetrigger via a labile, cleavable, or reversible bond or linker.

A targeting group can enhance the pharmacokinetic or biodistributionproperties of an RNAi trigger or conjugate to which it is attached toimprove cell- or tissue-specific distribution and cell-specific uptakeof the conjugate. In some instances, binding of a targeting group to acell or cell receptor may initiate endocytosis. A targeting group can bemonovalent, divalent, trivalent, tetravalent, or have higher valency.Representative targeting groups include, without limitation, compoundswith affinity to cell surface molecule, cell receptor ligands, hapten,antibodies, monoclonal antibodies, antibody fragments, and antibodymimics with affinity to cell surface molecules.

The RNAi trigger molecules described herein may be synthesized having areactive group, such as an amine group, at the 5′-terminus. The reactivegroup may be used to subsequently attach a targeting moiety usingmethods typical in the art.

In some embodiments, an Hif2α RNAi trigger includes a linking groupconjugated to the trigger. The linking group facilitates covalentlinkage of the trigger to a targeting group or delivery polymer ordelivery vehicle. The linking group can be linked to the 3′ or the 5′end of the RNAi trigger sense strand or antisense strand. In someembodiments, the linking group is linked to the RNAi trigger sensestrand. In some embodiments, the linking group is conjugated to the 5′or 3′ end of an RNAi trigger sense strand. In some embodiments a linkinggroup is conjugated to the 5′ end of an RNAi trigger sense strand.Examples of linking groups, include, but are not limited to:Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me,reactive groups such a primary amines and alkynes, alkyl groups, abasicribose, ribitol, and/or PEG groups.

A linker or linking group is a connection between two atoms that linksone chemical group (such as an RNAi trigger) or segment of interest toanother chemical group (such as a targeting group or delivery polymer)or segment of interest via one or more covalent bonds. A labile linkagecontains a labile bond. A linkage may optionally include a spacer thatincreases the distance between the two joined atoms. A spacer mayfurther add flexibility and/or length to the linkage. Spacers mayinclude, but are not be limited to, alkyl groups, alkenyl groups,alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, andaralkynyl groups; each of which can contain one or more heteroatoms,heterocycles, amino acids, nucleotides, and saccharides. Spacer groupsare well known in the art and the preceding list is not meant to limitthe scope of the description.

Targeting groups and linking groups include, but are not limited to,(Alk-C6), (Alk-C6-C6), (Alk-C6-SMPT-C6), (Alk-PEG5-C6),(Alk-PEG5-C6)(Alk-PEG5-Ser), (Alk-SMPT-C6), (Alk-SS-C6), (C6-SS-Alk-Me),(Chol-TEG), (DBCO-TEG), (Me-Alk-SS-C6), (NAG13), (NH2-C6). In someembodiments, any of the Hif2α RNAi trigger sense strands listed in Table2B which contains a 3′ or 5′ targeting group or linking group, mayalternatively contain no 3′ or 5′ targeting group or linking group, ormay contain a different 3′ or 5′ targeting group or linking groupincluding, but not limited to, those depicted in Table 4.

In some of the targeting group and linking group structures shown inTable 4, the RNAi trigger is shown and denoted by Trigger, RNA, R, or R1or R2 (i.e. Trigger, RNA or R1 or R2 each comprises the RNAi trigger).For example, with respect to (Alk-C6-Ser), (Alk-PEG5-Ser), and(Alk-PEG13-Ser), one of R1 and R2 comprises the RNAi trigger and theother can be a hydrogen.

TABLE 4 Structures representing targeting groups and linking groups.

In some embodiments, a delivery vehicle may be used to deliver an RNAitrigger to a cell or tissue. A delivery vehicle is a compound thatimproves delivery of the RNAi trigger to a cell or tissue. A deliveryvehicle can include, or consist of, but is not limited to: a polymer,such as an amphipathic polymer, a membrane active polymer, a peptide, amelittin peptide, a melittin-like peptide, a lipid, a reversiblymodified polymer or peptide, or a reversibly modified membrane activepolyamine.

In some embodiments, the RNAi triggers can be combined with lipids,nanoparticles, polymers, liposomes, micelles, DPCs or other deliverysystems available in the art. The RNAi triggers can also be chemicallyconjugated to targeting groups, lipids (including, but not limited tocholesterol and cholesteryl derivatives), nanoparticles, polymers,liposomes, micelles, DPCs (see, for example WO 2000/053722, WO2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO2013/158141, each of which is incorporated herein by reference), orother delivery systems available in the art.

In some embodiments, pharmaceutical compositions for delivering an Hif2αRNAi trigger to a tumor cell in vivo are described. Such pharmaceuticalcompositions can include, but are not limited to, an Hif2α RNAi triggerconjugated to delivery polymer to form an RNAi trigger-delivery polymerconjugate. In some embodiments, the delivery polymer is a membraneactive polyamine. In some embodiments, the delivery polymer is areversibly modified membrane active polyamine.

Hif2α RNAi Trigger-Delivery Polymer Conjugates

In some embodiments, we describe compositions represented by theformula:

wherein RNAi trigger is an Hif2α RNAi trigger as described herein, P isa membrane active polyamine, M¹ comprises a targeting group linked to Pvia reversible physiologically labile linkage L¹, and M² comprises asteric stabilizer linked to P via reversible physiologically labilelinkage L², x is greater than 1, y is greater than or equal to 0.(M²-L²)_(y)-P-(L¹-M¹)_(x), is not membrane active. As used herein,(M²-L²)_(y)-P-(L¹-M¹)_(x) refers to a delivery polymer. Cleavage of(L¹-M¹) and (M²-L²) restores P to a membrane active state. In someembodiments, the value of x+y is greater than 80%, greater than 90%, orgreater than 95% of the number of primary amines of P. In someembodiments, the value of x+y is greater than 80%, greater than 90%, orgreater than 95% of the number of primary amines on a population of P.The value of n can be from 0.25 to 5 (one (1) RNAi trigger per every 4polymers to 5 RNAi triggers per polymer). In some embodiments, the valueof n is 0.5 to 5. In some embodiments, n is 0.5-2. In some embodiments,n is 0.8-1.6. In some embodiments, x is 1-20, 2-20, 3-20, 4-20, 5-20,6-20,7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20.

In some embodiments, M¹ comprises an integrin-binding compound. In someembodiments, the integrin-binding compound comprises an α_(v)β₃-bindingcompound. In some embodiments, the integrin-binding compound comprisesan RGD ligand. In some embodiments, the α_(v)β₃-binding compoundcomprises an RGD ligand. In some embodiments the RGD ligand comprises anRGD mimic. In some embodiments, the steric stabilizer comprises apolyethylene glycol (PEG). In some embodiments, cleavage of L¹ and/or L²restores an unmodified amine on P. In some embodiments, (L¹-M¹) and(L²-M²) are independently tetrapeptide modifying agents and/or dipeptidemodifying agents. In some embodiments, L¹ and L² are independentlytetrapeptide linkages or dipeptide-PABC (p-amidobenzyl-carbamate)linkages. In some embodiments, L¹ and L² are tetrapeptide linkages. Inother embodiments, L¹ and L² are dipeptide-PABC linkages. In someembodiments, L¹ is a dipeptide-PABC linkage and L² is a tetrapeptidelinkage. In other embodiments, L¹ is a tetrapeptide linkage and L² is adipeptide-PABC linkage. In some embodiments, a tetrapeptide linkage isan FCitFP (Phenylalanine-Citrulline-Phenylalanine-Proline) tetrapeptidelinkage. In some embodiments, a dipeptide-PABC linkage is an ACit-PABClinkage. For x=2 or more, L can be all tetrapeptide linkages, alldipeptide-PABC linkages, or a combination tetrapeptide linkages anddipeptide-PABC linkages. For y=2 or more, L² and be all tetrapeptidelinkages, all dipeptide-PABC linkages, or a combination tetrapeptidelinkages and dipeptide-PABC linkages.

In some embodiments, a described Hif2α RNAi trigger is conjugated to areversibly modified membrane active polyamine to form an RNAitrigger-delivery polymer conjugate. In some embodiments, the RNAitrigger-delivery polymer conjugate comprises the formula represented by:

(RNAi trigger)_(n)-poly(A_(a)-co-(B_(b)-graft-(C_(c);D_(d))))  (formula2)

-   -   wherein    -   A is a hydrophobic group-containing monomeric unit,    -   B is a primary amine-containing monomeric unit,    -   C comprises an integrin-binding ligand linked (i.e., grafted) to        a primary amine-containing monomeric unit via a reversible        physiologically labile linkage,    -   D comprises a steric stabilizer linked (i.e., grafted) to a        primary amine-containing monomeric unit via a reversible        physiologically labile linkage,    -   a is an integer greater than zero,    -   b is an integer greater than or equal to two,    -   c is an integer greater than or equal to one,    -   d is an integer greater than or equal to one,    -   the value of c+d is greater than 80%, greater than 85%, greater        than 90%, or greater than 95% of the value of b,    -   poly(A_(a)-co-B_(b)) is a membrane active polyamine copolymer        having A and B monomeric units RNAi trigger comprises a Hif2α        RNAi trigger described herein, and n has a value from 0.25        (i.e., conjugated to only one out of every four delivery        polymers) to 5.0.

Poly(A_(a)-c(B_(b)-graft-(C_(c); D_(d)))) is not membrane active. Insome embodiments, the integrin-binding compound comprises anα_(v)β₃-binding compound. In some embodiments, the integrin-bindingcompound comprises an RGD ligand, such as an RGD mimic. In someembodiments, the α_(v)β₃-binding compound comprises an RGD ligand, suchas an RGD mimic. In some embodiments, the steric stabilizer comprises apolyethylene glycol (PEG). In some embodiments, the PEG contains 2 to 25ethylene glycol units. In some embodiments, c is any integer from 1-75,1-50, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 10-35, 10-30, 10-25, 10-20, or15-20. In some embodiments, n has a value from 0.5 to 2. In someembodiments, the ratio A:B (i.e., a:b) is 30:70 to 60:40. In someembodiments, the ratio A:B is 60:40 to 40:60. In some embodiments, theratio A:B is about 45+5:55+5. In some embodiments, the ratio A:B isabout 44:56. In some embodiments, the ratio A:B is about 46:54. In someembodiments, the molecular weight (Mw) of the polymer is 30 kDa-70 kDa.In other embodiments, the Mw of the polymer is 40 kDa-60 kDa. In otherembodiments, the Mw of the polymer is 40 kDa-50 kDa. In yet otherembodiments, the Mw of the polymer about 43 kDa to about 48 kDa. In someembodiments, the polymer has a polydispersity index (PDI) less than 1.4,less than 1.3, 1.25, less than 1.2, less than 1.15, or less than 1.1. Insome embodiments, the polymer contains a terminal azide group forattachment of an RNAi trigger. In some embodiments, n is 0.8-1.6. Insome embodiments, n is 1+0.5. In some embodiments, c is 1-20, 2-20,3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20,14-20, or 15-20. In some embodiments, the value of c+d is greater than80%, greater than 90%, or greater than 95% of the value of b. In someembodiments, C is RGD-PEG_(x)-FcitFPro and D is PEG_(y)-ACit-PABC,wherein x is 1-50, y is 4-30. In some embodiments, x is greater than y.

In some embodiments, polyamine poly(A_(a)-co-B_(b)) is a poly(acrylate)random copolymer wherein A is a hydrophobic group-containing acrylatemonomer and B is a primary amine-containing acrylate monomer. In someembodiments A is a propyl acrylate monomer and B is an ethoxy-ethylamineacrylate monomer.

Membrane active polyamines are membrane active and therefore capable ofdisrupting plasma membranes or lysosomal/endocytic membranes. As usedherein, membrane active polyamines are surface active, amphipathicpolymers that are able to induce one or more of the following effectsupon a biological membrane: an alteration or disruption of the membranethat allows non-membrane permeable molecules to enter a cell or crossthe membrane, pore formation in the membrane, fission of membranes, ordisruption or dissolving of the membrane. As used herein, a membrane, orcell membrane, comprises a lipid bilayer. The alteration or disruptionof the membrane can be functionally defined by the peptide's activity inat least one the following assays: red blood cell lysis (hemolysis),liposome leakage, liposome fusion, cell fusion, cell lysis, andendosomal release. Peptides, or modified peptides that preferentiallycause disruption of endosomes or lysosomes over plasma membranes areconsidered endosomolytic. A reversibly modified membrane activepolyamine is an example of an endosomolytic peptide. The effect ofmembrane active polymers on a cell membrane may be transient. Membraneactive polymers possess affinity for the membrane and cause adenaturation or deformation of bilayer structures. Delivery of a RNAitrigger to a cell is mediated by the membrane active polyaminedisrupting or destabilizing the plasma membrane or an internal vesiclemembrane (such as an endosome or lysosome), including forming a pore inthe membrane, or disrupting endosomal or lysosomal vesicles therebypermitting release of the contents of the vesicle into the cellcytoplasm. A preferred polymer is an amphipathic poly(acrylate) randomcopolymer.

Integrin-Binding Compound

An integrin-binding compound has affinity for one or more integrinsexpressed on a cell surface. A non-limiting example of an integrinincludes an α_(v)β₃ integrin. Examples of integrin-binding compoundsinclude, but are not limited to: α_(v)β₃-binding compounds, RGD ligand.RGD ligands include RGD peptide-containing compounds and RGDmimic-containing compounds. As used herein, an RGD peptide comprises anarginine-glycine-aspartate tripeptide. An RGD peptide may beconformationally constrained. An RGD peptide may have non-peptidecomponents linked to the RGD amino acid sequence.

As used herein, an RGD ligand comprises an RGD peptide or RGD mimic<1500kDa in size that binds to (has affinity for) an integrin, such as analpha v/beta 3 (αvβ3 or α_(v)β₃) integrin.

As used herein, an RGD mimic is a non-peptide synthetic molecule otherthan an RDG peptide that biologically mimics the active determinants ofan RGD peptide, an integrin-binding RGD portion of an integrin-bindingprotein, or an α_(v)β₃ integrin-binding RGD motif. An RGD mimic maycontain one or two naturally occurring amino acids linked via amidebonds. An RGD mimetic may be a modified peptide, contain non-standardamino acids or non-standard amino acid side chains.

In one embodiment, an RGD ligand comprises a guanidinium group linked toa glycine-aspartate dipeptide via an amide bond. Guanidinium groups ofthe invention have the structure represented by:

wherein R⁹ and R¹⁰ are independently hydrogen or alkyl and may byconnected to form a ring, and R¹¹ is a linker connecting the guanidiniumgroup to the glycine-aspartate dipeptide. The guanidinium group includesboth the structure represented above and its resonance structures. Apreferred linker is: —(CRR′)—(CRR′HCRR′)— or—(CRR′)—(CRR′)—(CRR′)—(CRR′)—, wherein: a) each R is independentlyoptional and if present is independently hydrogen, alkyl, or aryl, b) R′is independently hydrogen, alkyl, aryl, or NH2, and c) each carbon (C)may be linked by single bonds, a single bond and a double bond, oraromatic bonds.

In some embodiments, an RGD mimic contains a phenoxy group attached tothe aspartate amino acid. In some embodiments, an RGD mimic comprises aquanidinium-glycine-aspartate-4-aminophenoxy compound. In someembodiments, a quanidinium-glycine-aspartate-4-aminophenoxy compoundcomprises the structure represented by:

wherein R^(D) is:

In some embodiments, a guanidinium is

and their resonance structures.

In some embodiments, an RGD mimic comprises the structure representedby:

-   -   wherein:        -   R¹⁴ is

and

A comprises a linker. The linker connects the RGD mimic to anothermolecule such as a dipeptide amidobenzyl-carbonate or tetrapeptide,provides for increased solubility, or provides a means for covalentlinkage to another molecule.

Steric Stabilizer

As used herein, a steric stabilizer is a non-ionic hydrophilic polymer(either natural, synthetic, or non-natural) that prevents or inhibitsintramolecular or intermolecular interactions of a polymer to which itis attached relative to the polymer containing no steric stabilizer. Asteric stabilizer hinders a polymer to which it is attached fromengaging in electrostatic interactions. Electrostatic interaction is thenon-covalent association of two or more substances due to attractiveforces between positive and negative charges. Steric stabilizers caninhibit interaction with blood components and therefore opsonization,phagocytosis, and uptake by the reticuloendothelial system. Stericstabilizers can thus increase circulation time of molecules to whichthey are attached. Steric stabilizers can also inhibit aggregation of apolymer. In some embodiments, a steric stabilizer is a polyethyleneglycol (PEG) or PEG derivative. In some embodiments, a PEG can haveabout 1-500 ethylene monomers or units. In some embodiments, the PEGcontains 2-25 ethylene units. In some embodiments, the PEG contains 4-30ethylene units. In some embodiments, PEG contains 5-24 ethylene units.In some embodiments, a PEG has a molecular weight average of about85-20,000 Daltons (Da). In some embodiments a PEG has a molecular weightof about 85-1000 Da. As used herein, steric stabilizers prevent orinhibit intramolecular or intermolecular interactions of a polymer towhich it is attached relative to the polymer containing no stericstabilizer in aqueous solution.

Reversible Physiologically Labile Linkages/Modifying Agents

A membrane active polyamine may be reversibly modified. Reversiblemodification can be accomplished through reversible attachment ofmodifying agents to primary amines of the membrane active polyamine.

In some embodiments, a reversible physiologically labile linkagecomprises a tetrapeptide linkage. In some embodiments, P-(L¹-M¹)_(x)and/or P-(L²-M²)_(y) (of formula 1) comprises:

R⁵-A⁴-A³-A²-A¹-R⁶

wherein

-   -   R⁵ comprises a targeting group (M) or a steric stabilizer (M²),    -   A⁴ is a natural, non-natural isomeric, or synthetic hydrophobic        L amino acid wherein the hydrophobicity index (Monera et al, J.        Protein Sci. 1995, 1, 319) at pH 7 is 41 or greater, normalized        to glycine, as it relates to the composition of the amino acid        side chain (R-group),    -   A³ is an uncharged hydrophilic L amino acid wherein the        hydrophobicity index (Monera et al, J. Protein Sci. 1995,        1, 319) at pH 7 is −28 or less, normalized to glycine, as it        relates to the composition of amino acid side chain (R-group),    -   A² is a natural, non-natural isomeric, or synthetic hydrophobic        L amino acid wherein the hydrophobicity index (Monera et al, J.        Protein Sci. 1995, 1, 319) at pH 7 is 41 or greater, normalized        to glycine, as it relates to the composition of the amino acid        side chain (R-group),    -   A¹ is L-proline, L-leucine, or L-N-methyl alanine, and    -   R⁶ is P, wherein P is a membrane active polyamine of formula 1.

In some embodiments, A¹ is proline, A² and A⁴ are independently alanine,valine, leucine, isoleucine or phenylalanine (side chains of —CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, or —CH₂C₆H₆, respectively), andA³ is citrulline or asparagine (side chains or —(CH₂)₃NHCONH₂ or—CH₂CONH₂, respectively).

In some embodiments, A¹ is proline, A² and A⁴ are phenylalanine, and A³is citrulline (FCitFPro). In some embodiments, A¹ is proline, A² isphenylalanine, and A³ is citrulline, and A⁴ is alanine (ACitFPro).

In some embodiments, a tetrapeptide modifying agent has the structurerepresented by:

-   -   wherein,    -   R⁵ comprises a targeting group (M) or a steric stabilizer (M²),    -   R4 is a side chain of a natural, non-natural isomeric, or        synthetic hydrophobic amino acid,    -   R³ is a side chain of an uncharged hydrophilic amino acid,        preferably citrulline,    -   R² is a side chain of a natural, non-natural isomeric, or        synthetic hydrophobic amino acid, preferably phenylalanine,    -   X and Y are:        -   a) (CH₂)₂(CH₃)₂ and H, respectively (tetrapeptide A′ is            Leucine),        -   b) CH₃— and CH₃—, respectively (tetrapeptide A′ is N-methyl            alanine), or        -   c) CH₂— and CH₂—CH₂—, respectively (tetrapeptide A′ is            proline); and    -   R is

Reaction of the tetrapeptide modifying agent with a polyamine yieldsP-(L-M).

In some embodiments, R4 is a side chain of phenylalanine or alanine. Insome embodiments, R³ is a side chain of citrulline. In some embodiments,R² is a side chain of phenylalanine.

In some embodiments, the membrane active polyamine is modified withdipeptide modifying agents (dipeptide-PABC-PNP modifying agent) havingthe general form:

R-A¹A²-amidobenzyl-carbonate.

wherein R comprises a steric stabilizer or targeting group, A¹ is ahydrophobic amino acid, and A² is a hydrophilic uncharged amino acid.Reaction of the modifying agent carbonate with a polymer amine yields acarbamate linkage. In some embodiments, the amidobenzyl group is ap-amidobenzyl group. In some embodiments, the carbonate is an activatedamine reactive carbonate. In some embodiments, dipeptide-PABC cleavablelinkers have the general structure:

wherein R4 comprises a targeting group or steric stabilizer, R3comprises an amine reactive carbonate moiety, such as a para-nitrophenylgroup, R1 is the side chain of a hydrophobic amino acid, such asPhenylalanine or Alanine and R2 is the side chain of a hydrophilicuncharged are amino acid, such as citrulline (Cit). In some embodiments,R1 is the side chain of Phenylalanine or Alanine. In some embodiments,R2 is the side chain of citrulline (Cit).

In some embodiments, an RGD modifying agent comprises the structurerepresented by:

wherein R¹⁴ is a guanidinium-containing group as defined above, A¹comprises a PEG-containing linker, R¹ is a side chain of a Phenylalanineor Alanine, R² is a side chain of citrulline, and R³ is anamine-reactive carbonate.

A delivery polymer can include a polyamine reversibly modified byreaction of primary amines on the polymer with a disubstitutedalkylmaleic anhydride:

wherein R1 comprises a targeting group or a steric stabilizer.

In some embodiments, the disubstituted alkylmaleic anhydride has thestructure represented by:

wherein R1 comprises an targeting group or a steric stabilizer.

In some embodiments, a targeting group (e.g., RGD ligand) is linked to amodifying agent via a linker, such as a PEG linker. The PEG linker canhave 1-50 ethylene units.

RGD and PEG modifying agents are shown in FIGS. 1-7.

In some embodiments, we describe compositions represented by theformula:

wherein: RNAi trigger is an Hif2α RNAi trigger, n is 0.5-5, P is amembrane active polyamine, L¹-M¹ comprises RGD-PEG_(a)-FCitFPro-, a is1-50, x is 1-20, L²-M² comprises PEG_(b)-ACit-PABC-, b is 4-30, and y isgreater than or equal to 0, and (M²-L²)_(y)-P-(L-M). is not membraneactive. In some embodiments, the value of x+y is greater than 80%,greater than 90%, or greater than 95% of the number of primary amines ofP. In some embodiments, n is 0.5-2. In some embodiments, n is 0.8-1.6.In some embodiments, x is 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20,9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20. In some embodiments,the value of a is greater than the value of b.

Pharmaceutical Compositions

In some embodiments, at least one of the described Hif2α RNAi triggersis used in the preparation of a pharmaceutical composition (i.e.,medicament) for treatment of a subject that would benefit from reductionor inhibition in Hif2α expression. These pharmaceutical compositions areuseful in the inhibition of the expression of the Hif2α gene in a cell,a tissue, or an organism. In some embodiments, the describedpharmaceutical compositions are used to treat a subject having a diseaseor disorder that would benefit from reduction or inhibition in Hif2αexpression.

As used herein, a pharmaceutical composition or medicament comprises apharmacologically effective amount of at least one of the describedHif2α RNAi triggers or Hif2α RNAi trigger-containing conjugates and oneor more pharmaceutically acceptable excipients. Pharmaceuticallyacceptable excipients (excipients) are substances other than the ActivePharmaceutical ingredient (API, therapeutic product, e.g., RNAi trigger)that have been appropriately evaluated for safety and are intentionallyincluded in the drug delivery system. Excipients do not exert or are notintended to exert a therapeutic effect at the intended dosage.Excipients may act to a) aid in processing of the drug delivery systemduring manufacture, b) protect, support or enhance stability,bioavailability or patient acceptability of the API, c) assist inproduct identification, and/or d) enhance any other attribute of theoverall safety, effectiveness, of delivery of the API during storage oruse. A pharmaceutically acceptable excipient may or may not be an inertsubstance.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, binders,buffering agents, carriers, coating agents, colors, delivery enhancers,dextran, dextrose, diluents, disintegrants, emulsifiers, extenders,fillers, flavors, glidants, humectants, lubricants, oils, polymers,preservatives, saline, salts, solvents, sugars, suspending agents,sustained release matrices, sweeteners, thickening agents, tonicityagents, vehicles, water-repelling agents, and wetting agents.

A pharmaceutical composition can contain other additional componentscommonly found in pharmaceutical compositions. Such additionalcomponents include, but are not limited to: anti-pruritics, astringents,local anesthetics, or anti-inflammatory agents (e.g., antihistamine,diphenhydramine, etc.). It is also envisioned that cells, tissues orisolated organs that express or comprise the herein defined RNAitriggers may be used as “pharmaceutical compositions”. As used herein,“pharmacologically effective amount,” “therapeutically effectiveamount,” or simply “effective amount” refers to that amount of an RNAitrigger to produce the intended pharmacological, therapeutic orpreventive result.

In some embodiments, a described Hif2α RNAi trigger is combined one ormore additional therapeutics or treatments including, but not limitedto: a second Hif2α RNAi trigger or other RNAi agent, a small moleculedrug, an antibody, an antibody fragment, and/or a vaccine.

The described RNAi triggers and pharmaceutical compositions comprisingHif2α RNAi triggers disclosed herein may be packaged or included in akit, container, pack, or dispenser. The Hif2α RNAi triggers andpharmaceutical compositions comprising said Hif2α RNAi triggers may bepackaged in pre-filled syringes or vials.

Cells, tissues, and non-human organisms that include at least one of theHif2α RNAi triggers described herein is contemplated. The cell, tissue,or non-human organism is made by delivering the RNAi trigger to thecell, tissue, or non-human organism by any means available in the art.In some embodiments, the cell is a mammalian cell, including, but nolimited to, a human cell. The cell, tissue, or non-human organisms areuseful for research or as research tools (e.g., drug testing ordiagnoses).

Method of Treatment

In some embodiments, the Hif2α RNAi triggers described herein are usedto treat a subject having a disease or disorder that would benefit fromreduction or inhibition in Hif2α expression. In some embodiments, thedescribed Hif2α RNAi triggers are used to treat or prevent at least onesymptom in a subject having a disease or disorder that would benefitfrom reduction or inhibition in Hif2α expression. The subject isadministered a therapeutically effective amount of any one or more ofthe described RNAi triggers thereby treating the symptom.

In some embodiments, the Hif2α RNAi triggers are used to treat or managea clinical presentation wherein a subject in need of such treatment,prevention or management is administered a therapeutically orprophylactically effective amount of one or more of the Hif2α RNAitriggers or Hif2α RNAi trigger-containing compositions described herein.In some embodiments, the method comprises administering a compositioncomprising an Hif2α RNAi trigger molecule described herein to a mammalto be treated.

Representative diseases that would benefit from a reduction and/orinhibition of Hif2α gene expression include, but are not limited to,cancer, renal cancer, clear cell renal cell carcinoma, non-small celllung cancer, astrocytoma (brain cancer), bladder cancer, breast cancer,chondrosarcoma, colorectal carcinoma, gastric carcinoma, glioblastoma,head and neck squamous cell carcinoma, hepatocellular carcinoma, lungadenocarcinoma, neuroblastoma, melanoma, multiple myeloma, ovariancancer, rectal cancer, metastases, gingivitis, psoriasis, Kaposi'ssarcoma-associated herpesvirus, preeclampsia, inflammation, chronicinflammation, neovascular diseases, and rheumatoid arthritis.

In some embodiments, an Hif2α RNAi trigger can be used to inhibitexpression of Hif2α in a cell, group of cells, or a tissue, e.g., in asubject. In some embodiments, an Hif2α RNAi trigger can be used toformulate a composition for inhibiting expression of Hif2α in a cell,group of cells, or a tissue, e.g., in a subject. In some embodiments, atherapeutically effective amount of one type (or several differenttypes) of Hif2α RNAi triggers as described herein is administered to asubject, thereby inhibiting expression of Hif2α in the subject (e.g., anamount effective to inhibit expression of Hif2α in the subject).

As used herein, the terms “silence,” “reduce,” “inhibit,”“down-regulate,” or “knockdown gene expression,” when referring to anHif2α gene, mean that the expression of the gene, as measured by thelevel of RNA transcribed from the gene or the level of polypeptide,protein, or protein subunit translated from the mRNA in a cell, group ofcells, or tissue, in which the Hif2α gene is transcribed, is reducedwhen the cell, group of cells, or tissue, is treated with the describedHif2α RNAi triggers as compared to a second cell, group of cells, ortissue that has or has not been so treated or compared to the same cell,group of cells, or tissue, prior to administration of the Hif2α RNAitrigger.

In some embodiments, the gene expression level and/or mRNA level ofHif2α in a subject to whom a described Hif2α RNAi trigger isadministered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%relative to the subject prior to being administered the Hif2α RNAitrigger or to a subject not receiving the Hif2α RNAi trigger. The geneexpression level and/or mRNA level in the subject may be reduced in acell, group of cells, and/or tissue of the subject. In some embodiments,the protein level of Hif2α in a subject to whom a described Hif2α RNAitrigger has been administered is reduced by at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% relative to the subject prior to being administered theHif2α RNAi trigger or to a subject not receiving the Hif2α RNAi trigger.The protein level in the subject may be reduced in a cell, group ofcells, tissue, blood, and/or other fluid of the subject. A reduction ingene expression, mRNA, or protein levels can be assessed by any methodsknown in the art. Reduction or decrease in Hif2α mRNA level and/orprotein level are collectively referred to herein as a reduction ordecrease in Hif2α or inhibiting or reducing the expression of Hif2α.

“Introducing into a cell”, when referring to an RNAi trigger, meansfunctionally delivering the RNAi trigger into a cell. By functionaldelivery, it is meant that the RNAi trigger is delivered to the cell andhas the expected biological activity, (e.g., sequence-specificinhibition of gene expression).

The route of administration is the path by which an RNAi trigger isbrought into contact with the body. In general, methods of administeringdrugs and nucleic acids for treatment of a subject are well known in theart and can be applied to administration of the compositions describedherein. The compounds described herein can be administered via anysuitable route in a preparation appropriately tailored to the particularroute. Thus, the compounds described herein can be administered byinjection, for example, intravenously, intramuscularly,intracutaneously, subcutaneously, or intraperitoneally.

In some embodiments, the Hif2α RNAi trigger molecules or compositionsdescribed herein can be delivered to a cell, group of cells, tissue, orsubject using oligonucleotide delivery technologies known in the art. Ingeneral, any suitable method recognized in the art for delivering anucleic acid molecule (in vitro or in vivo) can be adapted for use withan Hif2α RNAi trigger described herein. For example, delivery can be bylocal administration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal),intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical(including buccal and sublingual) administration, In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

The above provided embodiments and items are now illustrated with thefollowing, non-limiting examples.

EXAMPLES Example 1. RNAi Trigger Synthesis

A) Synthesis. RNAi trigger molecules were synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis. Depending on the scale either a MerMade96E (Bioautomation) ora MerMade12 (Bioautomation) was used. Syntheses were performed on asolid support made of controlled pore glass (CPG, 500 Å or 600 Å,obtained from Prime Synthesis, Aston, Pa., USA). All DNA, 2′-modifiedRNA, and UNA phosphoramidites were purchased from Termo FisherScientific (Milwaukee, Wis., USA). Specifically, the following2′-O-Methyl phosphoramidites were used:(5′-O-dimethoxytrityl-N⁶-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropy-lamino)phosphoramidite,5′-O-dimethoxy-trityl-N⁴-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite,(5′-O-dimethoxytrityl-N²-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite,and5′-O-dimethoxy-trityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite.The 2′-Deoxy-2′-fluoro-phosphor-amidites carried the same protectinggroups as the 2′-O-methyl RNA amidites. The following UNAphosphoramidites were used:5′-(4,4′-Dimethoxytrityl)-N-benzoyl-2′,3′-seco-adenosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphor-amidite,5′-(4,4′-Dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-(4,4′-Dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and5′-(4,4′-Dimethoxy-trityl)-2′,3′-seco-uridine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. Allamidites were dissolved in anhydrous acetonitrile (50 mM) and molecularsieves (3 Å) were added. In order to introduce the TEG-Cholesterol atthe 5′-end of the oligomers, the1-Dimethoxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramiditefrom Glen Research (Sterling, Va., USA) was employed. The5′-modifications were introduced without any modification of thesynthesis cycle. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile)was used as activator solution.

Coupling times were 10 min (RNA), 180 sec (Cholesterol), 90 sec (2′OMeand UNA), and 60 sec (2′F and DNA). In order to introducephosphorothioate linkages, a 100 mM solution of 3-phenyl1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster,Mass., USA) in anhydrous Acetonitrile was employed. See Tables 1-2 and 5for specific sequences.

B. Cleavage and deprotection of support bound oligomer. Afterfinalization of the solid phase synthesis, the dried solid support wastreated with a 1:1 volume solution of 40 wt. % methylamine in water and28% ammonium hydroxide solution (Aldrich) for two hours at 30° C. Thesolution was evaporated and the solid residue was reconstituted in water(see below).

C. Purification. Crude Cholesterol containing oligomers were purified byreverse phase HPLC using a Waters XBridge BEH300 C4 5u Prep column and aShimadzu LC-8 system. Buffer A was 100 mM TEAA, pH 7.5 and contained 5%Acetonitrile and buffer B was 100 mM TEAA and contained 95%Acetonitrile. UV traces at 260 nm were recorded. Appropriate fractionswere then run on size exclusion HPLC using a GE Healthcare XK 16/40column packed with Sephadex G-25 medium with a running buffer of 100 mMammonium bicarbonate, pH 6.7 and 20% Acetonitrile. Other crude oligomerswere purified by anionic exchange HPLC using a TKSgel SuperQ-5PW 13ucolumn and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH9.0 and contained 20% Acetonitrile and buffer B was the same as buffer Awith the addition of 1.5 M sodium chloride. UV traces at 260 nm wererecorded. Appropriate fractions were pooled then run on size exclusionHPLC as described for cholesterol containing oligomers.

D. Annealing. Complementary strands were mixed by combining equimolarsolutions (sense and antisense) in 0.2×PBS (Phosphate-Buffered Saline,1×, Corning, Cellgro) to form the RNAi triggers. This solution wasplaced into a thermomixer at 70° C., heated to 95° C., held at 95° C.for 5 min, and cooled to room temperature slowly. Some RNAi triggerswere lyophilized and stored at −15 to −25° C. Duplex concentration wasdetermined by measuring the solution absorbance on a UV-Vis spectrometerin 0.2×PBS. The solution absorbance at 260 nm was then multiplied by aconversion factor and the dilution factor to determine the duplexconcentration. Unless otherwise stated, all conversion factor was 0.037mg/(mL cm). For some experiments, a conversion factor was calculatedfrom an experimentally determined extinction coefficient.

Example 2. Synthesis of APN 1170-100A (100A). APN 1203-006 (006). APN1203-064 (064) Amphipathic Membrane Active Polyamines

MW Theoretical MW % Amine % Alkyl % End Group Azides/ Polymer(protected) (deprotected) PDI Incorp. Incorp. Removal Polymer APN1170-100A 64,430 45,765 1.22 56 44 0 1.25 APN 1203-006 60,330 43,5781.05 56 44 99 1.14 APN 1203-062 65,170 46,736 1.05 54 46 99 0.96

A. Materials. 2,2′-Azobis(2,4-dimethyl valeronitrile) (V-65, radicalinitiator) was purchased from Wako Pure Chemical Industries. Propylacrylate was purchased from Polysciences Inc. N-Boc-ethoxy-ethylamineacrylate was obtained from WuXi Inc.2-(Dodecylthio-carbonothioylthio)-2-methylpropionic acid (DDMAT, RAFTAgent), 1,1′-Azobis-(cyclo-hexanecarbonitrile) (ACHN), 1-Ethylpiperidinehypophosphite (EPHP), Pentafluorophenol, N,N′-Dicyclohexylcarbodiimideand N,N-diisopropyl-ethylamine were purchased from Sigma Aldrich.O-(2-Aminoethyl)-O′-(2-azidoethyl)triethylene Glycol (azido-PEG₄-amine)was purchased from Biomatrik Inc.

B. RAFT copolymer of N-Boc-ethoxyethylamine acrylate and propyl acrylate(EAP). Solutions of V-65 (2 mg/mL) and RAFT agent DDMAT (10 mg/mL) inbutyl acetate were prepared. Monomer molar feed was 52%N-Boc-ethoxyethylamine acrylate, 48% propyl acrylate. Theoretical Mw was75,000. RAFT agent (DDMAT) to initiator (V-65) molar ratio was 6.67:1.

N-Boc-ethoxyethylamine acrylate (1.778 g, 6.86 mmol), propyl acrylate(0.794 mL, 0.722 g, 6.33 mmol), DDMAT solution (1.215 mL, 0.0333 mmol),V-65 solution (0.621 mL, 0.005 mmol), and butyl acetate (10.2 mL) wereadded to a 20 mL glass vial with a stir bar. The vial was sealed with asepta cap and the solution bubbled with nitrogen using a long needlewith a second needle as the outlet for 1 h. The needles were removed andthe vial was heated to 50° C. for 24 h with stirring. The solution wasallowed to cool to room temperature and transferred equally between two50 mL centrifuge tube before hexane (35 mL) was added to both tubes. Thesolution was centrifuged for 2 min at 4400 rpm. The supernatant layerwas carefully decanted and the bottom layer rinsed with hexane. Thebottom layer of each tube was then re-dissolved in dichloromethane (7mL), precipitated in hexane (40 mL) and centrifuged once more. Thesupernatant was decanted and the bottom layer rinsed with hexane beforethe layers were combined to one 50 mL centrifuge tube and the polymerwas dried under reduced pressure for several hours. The yield of crudeEAP copolymer was 2.1 g. Samples of the copolymer were taken formulti-angle light scattering (MALS), and ¹H-NMR.

-   -   Polymer 006: The composition determined by ¹H-NMR was 55%        N-Boc-ethoxyethylamine acrylate and 45% propyl acrylate. The Mw        for 006 determined by MALS was 58,600 g/mol with a        polydispersity index (PDI) of 1.04.    -   Polymer 100A: Composition by 1H-NMR: 56% N-Boc-ethoxyethylamine        acrylate and 44% propyl acrylate. MW by MALS: 65,150, PDI of        1.122.    -   Polymer 064: Composition by 1H-NMR: 54% N-Boc-ethoxyethylamine        acrylate and 46% propyl acrylate. The Mw for 064 determined by        MALS was 57,957 g/mol with a polydispersity index (PDI) of 1.07.

C. Radical induced w-end group removal (polymers 006 and 064). Solutionsof 1,1′-Azobis-(cyclohexanecarbonitrile) (ACHN, 20 mg/mL) and1-Ethylpiperidine hypophosphite (EPHP, 100 mg/mL) were prepared intoluene. EAP (2 g, 0.035 mmol), ACHN (0.213 mL, 0.5 eq, 0.0174 mmol),EPHP (1.25 mL, 20 eq, 0.697 mmol), and toluene (25.2 mL) were added to a40 mL glass vial with a stir bar. The vial was sealed with a septa capand the solution bubbled with nitrogen using a long needle with a secondneedle as the outlet for 1 h. The needles were removed and the vial washeated to 100° C. for 2 h. The solution was allowed to cool to roomtemperature and −20 mL toluene was removed by rotary evaporation. Theremaining solution was transferred to a 50 mL centrifuge vial, andhexane (35 mL) was added. The solution was centrifuged for 2 min at 4400rpm. The supernatant layer was carefully decanted and the bottom layerrinsed with hexane. The bottom layer was then re-dissolved indichloromethane (7 mL), precipitated in hexane (40 mL) and centrifugedonce more. The supernatant was decanted and the bottom layer rinsed withhexane before the polymer was dried under reduced pressure for ˜1 h. Thepolymer was dissolved in methyl tert-butyl ether (80 mL) and transferredto a separatory funnel. The solution was then washed with 3×30 mLvolumes of H₂O followed by 3×30 mL volumes of saturated NaCl. Thepolymer solution was then dried over sodium sulfate, and vacuum filteredthrough 0.45 μm GHP filters. MTBE was removed via rotary evaporation andhigh vacuum. A sample was taken for monitoring of end group removalusing a UV spectrophotometer. End group removal was calculated to be99%. Samples were taken for MALS, GC-FID, and ¹H-NMR. The composition of006 by H-NMR was 55% N-Boc-eth oxyethylamine acrylate and 45% propylacrylate. The conversion of 006 determined by GC-FID was 81.4% for theN-Boc-ethoxyethylamine acrylate and 77.3% for the propyl acrylate. Theconversion of 100A determined by GC-FID conversion was 87% forN-Boc-ethoxyethylamine acrylate and 83% for propyl acrylate. The Mw forpolymer 006 determined by MALS was 57,700 g/mol with a polydispersityindex (PDI) of 1.06.

D. Pentafluorophenol activation of α-end group. EAP polymer (2 g, 0.0347mmol), pentafluorophenol (63.8 mg, 0.3466 mmol),N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol), anddichloromethane (40 mL) were added to a 100 mL round bottom flask with astir bar. The flask was stoppered with a rubber septum and the systemwas purged with nitrogen for 15 min. The solution was stirred for 16 hat room temperature. Additional Pentafluorophenol (63.8 mg, 0.3466 mmol)and N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol) were added, theflask stoppered with a rubber septum, and the system was purged withnitrogen for 15 min. The solution was stirred for 3 h at roomtemperature. The polymer was precipitated with hexane (˜10× volume),centrifuged, and the solvent was decanted. The polymer was dissolved inminimal dichloromethane, precipitated with hexane (˜10× volume),centrifuged, and the solvent was decanted. The polymer was dissolved inminimal ethyl acetate, precipitated with hexane (˜10× volume),centrifuged, and the solvent was decanted. The polymer precipitate wasdried under high vacuum until the solid reached a constant weight.

E. Azide functionalization of α-end group. In a 100 ml round bottomflask equipped with a rubber septum and stir bar, polymer from theprevious step (1.9 g, 0.0329 mmol) was dissolved in dichloromethane (38mL). Azido-PEG₄-Amine (86.4 mg, 0.3293 mmol) andN,N-Diisopropylethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added tothe flask with stirring. The system was purged with nitrogen for 15 min,and the reaction was left to stir at room temperature overnightAdditional Azido PEG₄ Amine (86.4 mg, 0.3293 mmol) andN,N-Diisopropylethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added tothe flask, the system was purged with N₂ gas, and the reaction wasstirred for 3 h at room temperature. The polymer was precipitated withhexane (˜10× volume), centrifuged, and the solvent was decanted. Thepolymer was dissolved in minimal dichloromethane, precipitated withhexane (˜10× volume), centrifuged, and the solvent was decanted. Thepolymer precipitate was dried under high vacuum until the solid reacheda constant weight. The yield of Azide functionalized EAP was 1.77 g.Samples of the copolymer were taken for multi-angle light scattering(MALS), and ¹H-NMR.

-   -   Polymer 006: The composition determined by ¹H-NMR was 56%        N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mw        determined by MALS was 60,330 g/mol with a polydispersity index        (PDI) of 1.05.    -   Polymer 100A: The composition by H-NMR was 56%        N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mw        determined by MALS: 64,430 with PDI of 1.217.    -   Polymer 064: The composition by 1H-NMR was 54%        N-Boc-ethoxyethylamine acrylate and 46% propyl acrylate. The Mw        determined by MALS: 65,170 with PDI of 1.05

Mono-Azide: The term “mono-azide” or “mono-azide polymer” indicates thatsteps D and E of the procedures above were done and an azide group wascoupled to the α-end group of the polymer.

F. Boc Deprotection and Tangential Flow Filtration. In a 100 mL roundbottom flask, 2M HCl in acetic acid (28 mL) was added to Azidefunctionalized EAP copolymer (1.67 g, 0.0277 mmol). The reaction wasstirred at room temperature for 1 h. De-ionized H₂O (56 mL) was added,and stirred for 10 min. The solution was then immediately exchanged with10 equivalent volumes of 5 mM Phosphate-Citrate buffer (pH 5) using amPES 30 kD 115 cm² filter module equipped with a tangential flowfiltration system (KrosFlo Research). The solution was then concentratedusing the apparatus to 55 mL final volume. A pH value of 5.1 wasrecorded. Samples were taken for concentration determination byheadspace gas chromatography. An aliquot was lyophilized and thenreconstituted in 33.3% Acetonitrile-d in Deuterium Oxide at aconcentration of 10 mg/mL for ¹H-NMR analysis. Theoretical MW wascalculated to be 43,026 g/mol 45,765 g/mol for 006 and 100Arespectively.

G. Using similar techniques, similar amphipathic membrane activepolyamines can be readily formed. Particularly, amphipathic membraneactive polyamines with molecular weight (Mw) 40-120 k protected (25 k to85 k deprotected), PDI ranges of 1.03 to 1.2, and monomer ratios of 35%amine monomer/65% hydrophobic group monomer to 70% amine monomer/30%hydrophobic group monomer.

Example 3. Synthesis of APN 1095-126 (126)

% End MW Theoretical MW % Amine % Alkyl Group Azides Per (protected)(deprotected) PDI Incorporation Incorporation Removal Polymer 66,67047,606 1.11 56 44 0 4.1

Synthesis of APN 1095-126 used dithiobenzoate moiety RAFT agent and AIBNRAFT initiator, compared to the trithiocarbonate moiety RAFT agent andV-65 RAFT initiator used for synthesis of 100A and 006. The conditionsfor this polymerization required different heating temperatures andtimes. In addition, this polymer required fractional precipitation. Thepolymer was not end capped, but the method of azide addition was thesame as 100A and 006.

A. Materials. Propyl acrylate was purchased from Polysciences Inc.N-Boc-ethoxyethylamine acrylate was obtained from WuXi Inc.4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPCPA, RAFT Agent),2,2′-Azobis(2-methylpropionitrile) (AIBN, radical initiator),Pentafluorophenol, N,N′-Dicyclohexylcarbodiimide andN,N-diisopropylethylamine were purchased from Sigma Aldrich.O-(2-Aminoethyl)-O′-(2-azidoethyl)triethylene Glycol (azido-PEG₄-amine)was purchased from Biomatrik Inc.

B. RAFT copolymer of N-Boc-ethoxyethylamine acrylate and propyl acrylate(EAP). The following procedure was repeated 8 times to yield a total of4.5513 g fractionated EAP copolymer. Solutions of AIBN (1.035 mg/mL) andRAFT agent CPCPA (50.54 mg/mL) in butyl acetate were prepared. Monomermolar feed was 52% N-Boc-ethoxyethylamine acrylate, 48% propyl acrylate.Theoretical Mw was 75,000. RAFT agent (CPCPA) to initiator (AIBN) molarratio was 6.67:1.

N-Boc-ethoxyethylamine acrylate (1.7879 g, 6.9 mmol), propyl acrylate(0.774 mL, 0.7121 g, 6.24 mmol), CPCPA solution (0.184 mL, 0.0333 mmol),AIBN solution (0.793 mL, 0.005 mmol), and butyl acetate (11.02 mL) wereadded to a 20 mL glass vial with a stir bar.

The vial was sealed with a septa cap and the solution bubbled withnitrogen using a long needle with a second needle as the outlet for 1 h.The needles were removed and the vial was heated to 50° C. for 24 h withstirring. The solution was allowed to cool to room temperature andtransferred to a 50 mL centrifuge tube before hexane (35 mL) was added.The solution was centrifuged for 2 min at 4400 rpm. The supernatantlayer was carefully decanted and the bottom layer rinsed with hexane.The bottom layer of each tube was then re-dissolved in dichloromethane(7 mL), precipitated in hexane (40 mL) and centrifuged once more. Thesupernatant was decanted and the bottom layer rinsed with Hexane beforethe polymer was dried under reduced pressure for several hours. Theyield of crude EAP copolymer was 1.734 g. Samples of the crude copolymerwere taken for multi-angle light scattering (MALS), and ¹H-NMR. Thedried, crude copolymer was dissolved in DCM (100 mg/mL). Hexane wasadded until just after the cloud point was reached. The resulting milkysolution was centrifuged. The bottom layer was extracted and fullyprecipitated into hexane. The fraction was centrifuged, after which thecopolymer was isolated and dried under vacuum. The yield of isolatedfraction of EAP copolymer was 0.602 g. Samples of the fractionatedcopolymer were taken for ¹H-NMR and MALS. The composition determined by¹H-NMR was 56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate.The Mw determined by MALS was 62,010 g/mol with a polydispersity index(PDI) of 1.14.

C. Pentafluorophenol activation of α-end group. EAP polymer (2 g, 0.0347mmol), pentafluorophenol (63.8 mg, 0.3466 mmol),N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol), anddichloromethane (40 mL) were added to a 100 mL round bottom flask with astir bar. The flask was stoppered with a rubber septum and the systemwas purged with nitrogen for 15 min. The solution was stirred for 16 hat room temperature. Additional Pentafluorophenol (63.8 mg, 0.3466 mmol)and N,N′-Dicyclohexylcarbodiimide (71.5 mg, 0.3466 mmol) were added, theflask stoppered with a rubber septum, and the system was purged withnitrogen for 15 min. The solution was stirred for 3 h at roomtemperature. The polymer was precipitated with hexane (˜10× volume),centrifuged, and the solvent was decanted. The polymer was dissolved inminimal dichloromethane, precipitated with hexane (˜10× volume),centrifuged, and the solvent was decanted. The polymer was dissolved inminimal ethyl acetate, precipitated with hexane (˜10× volume),centrifuged, and the solvent was decanted. The polymer precipitate wasdried under high vacuum until the solid reached a constant weight.

D. Azide functionalization of α-end group. In a 100 ml round bottomflask equipped with a rubber septum and stir bar, polymer from theprevious step (1.9 g, 0.0329 mmol) was dissolved in dichloromethane (38mL). Azido-PEG₄-Amine (86.4 mg, 0.3293 mmol) andN,N-Diisopropyl-ethylamine (46.8 mg, 63.1 μL, 0.3622 mmol) were added tothe flask with stirring.

The system was purged with nitrogen for 15 min, and the reaction wasleft to stir at room temperature overnight. Additional Azido PEG₄ Amine(86.4 mg, 0.3293 mmol) and N,N-Diisopropyl-ethylamine (46.8 mg, 63.1 μL,0.3622 mmol) were added to the flask, the system was purged with N₂ gas,and the reaction was stirred for 3 h at room temperature. The polymerwas precipitated with hexane (˜10× volume), centrifuged, and the solventwas decanted. The polymer was dissolved in minimal dichloromethane,precipitated with hexane (˜10× volume), centrifuged, and the solvent wasdecanted. The polymer precipitate was dried under high vacuum until thesolid reached a constant weight. The yield of Azide functionalized EAPwas 1.77 g. Samples of the copolymer were taken for multi-angle lightscattering (MALS), and ¹H-NMR. The composition determined by ¹H-NMR was56% N-Boc-ethoxyethylamine acrylate and 44% propyl acrylate. The Mwdetermined by MALS was 66,670 g/mol with a polydispersity index (PDI) of1.11.

E. Boc Deprotection and Tangential Flow Filtration. In a 100 mL roundbottom flask, 2M HCl in acetic acid (28 mL) was added to Azidefunctionalized EAP copolymer (1.67 g, 0.0277 mmol). The reaction wasstirred at room temperature for 1 hour. De-ionized H₂O (56 mL) wasadded, and stirred for 10 min. The solution was then immediatelyexchanged with 10 equivalent volumes of 5 mM Phosphate-Citrate buffer(pH 5) using a mPES 30 kD 115 cm² filter module equipped with atangential flow filtration system (KrosFlo Research). The solution wasthen concentrated using the apparatus to 55 mL final volume. A pH valueof 5.1 was recorded. Samples were taken for concentration determinationby headspace gas chromatography. An aliquot was lyophilized and thenreconstituted in 33.3% Acetonitrile-d in Deuterium Oxide at aconcentration of 10 mg/mL for ¹H-NMR analysis. Theoretical MW wascalculated to be 43,026 g/mol.

Example 4. Polymer Analytics

-   -   (i) MALS analysis. Approximately 10 mg of the copolymer was        dissolved in 0.5 mL 75% dichloromethane, 20% tetrahydrofiran,        and 5% acetonitrile. The molecular weight and polydispersity        (PDI) were measured using a Wyatt Heleos II multiangle light        scattering detector attached to a Shimadzu Prominence HPLC using        a Jordi 5 μm 7.8×300 Mixed Bed LS DVB column. Molecular weight        (polymer 006) before de-protection: 60,330 (PDI 1.05).    -   (ii) Monomer conversion by Gas Chromatography. Approximately 40        μL of copolymer solution (section B) was taken after mixing        (pre-N₂ bubbling), after N₂ bubbling, and after reaction        completion. Samples were diluted 100 fold into ethyl acetate.        The samples were analyzed with a Shimadzu GC-2010 plus equipped        with a flame ionization detector using a Phenomenex Zebron        capillary column (ZB-5, 30 m, 0.25 mm ID, 0.5 μm film        thickness). Using the pre-N₂ bubbled sample as a single point        calibration, monomer conversion was measured by comparing post        reaction monomer concentrations with pre reaction/post N₂        bubbling monomer concentrations.    -   (iii) Polymer concentration by propanol content using headspace        gas chromatography (HS-GC). Deprotected polymer solution (˜20        mg/mL) was diluted 50 fold into 3M NaOH using 1-Butanol as an        internal standard. The reaction tube was sealed and shaken for        1 h. The reaction was then incubated for at least 6 h at room        temperature. In a 10 mL headspace vial, hydrolyzed test article        (250 μL) was added to saturated NaCl (500 μL) and HCl (4M, 250        μL) and the system was sealed. Test articles were analyzed using        a Shimadzu GC-2010 plus with HS-20 headspace sampler using a        Phenomenex ZB-WAX plus gc column (30.0 m, 0.25 mm ID, 0.25 μm        film thickness). Propanol concentration was then quantitated        using an external standard curve or propanol containing the same        NaCl/HCl/NaOH matrix. Polymer concentration was then calculated        by dividing propanol concentration by the amount of propanol per        polymer as determined by monomer incorporation.    -   (iv) Azide quantitation using UV Spectroscopy. Deprotected        polymer solution (˜20 mg/mL) was diluted to −5 mg/mL in 60 mM        MES, pH 6. The polymer was then reacted with DBCO-amine (2.5        molar eq.) at room temperature for at least 6 h. The difference        in absorbance at 310 nm was calculated and azide content per        polymer was determined.

Example 5. Tangential Flow Filtration and Analysis of Conjugate

Following Conjugate formation, i.e., modification of polymer by additionof RGD and PEG modifying agents and attachment of RNAi trigger (seeexample 9 below) the conjugate solution (2 mg/mL, 10 mL) was exchangedwith 10 equivalent volumes of 10 mM Phosphate-Citrate buffer (pH 5)using a mPES 30 kD 20 cm² filter module equipped with a tangential flowfiltration system (KrosFlo Research). A pH value of 5.1 was recorded.

A. Conjugate characterization and analysis.

-   -   (i) Polymer Concentration Throughout Conjugation. The Same        Method as Section G(iii) was used throughout the assembly of the        conjugate to monitor polymer concentration.    -   (ii) Impurity quantitation by HPLC-reverse-phase chromatography.        Polymer conjugate (after TFF purification) was diluted to 1        mg/mL with H₂O and injected onto a Shimadzu Prominence HPLC with        a SPD-20A UV detector and a Waters Xbridge C18 5 μm 4.6×250 mm        column. The method used a binary gradient consisting of        H₂O/Acetonitrile/0.1% formic acid with detection set to 247 nm.        Concentrations of PEG_(n)-ACit-PABOH, RGD-PEG_(n)-FCFP-COOH, and        PNP were calculated using external standard quantitation.    -   (iii) RGD-PEG_(n)-FCitFP-TFP and PEG_(n)-ACit-PABC-PNP        modification through amino acid analysis. Polymer conjugate        (after TFF purification) with NorValine as an internal standard        was hydrolyzed for 16 h in HCl (6 M) at 110° C. in a sealed        hydrolysis tube. The hydrolysis solution was then neutralized        with NaOH, diluted with borate buffer (pH 10.1), and derivatized        with phthaldialdehyde/3-mercaptopropionic acid. The sample was        then injected onto a Shimadzu Nexera HPLC system with SIL-30A        autosampler, SPD-20A photo diode array detector, and a Waters        Xbridge C18 5 μm 4.6×250 mm column. Sample was eluted using a 10        mM Sodium tetraborate decahydrate/10 mM dibasic sodium        phosphate/5 mM Sodium azide and 45% Methanol/45%        Acetonitrile/10% H₂O binary gradient. UV detection was set to        338 nm. Alanine and Phenylalanine concentrations were calculated        using external standard curves. Alanine and Phenylalanine        concentrations along with polymer concentration and monomer        incorporations were used to calculate total amine group        modification, as well as the ratio between both ligands.    -   (iv) RNA trigger quantitation and conjugate purity by size        exclusion chromatography. A Shimadzu Prominence HPLC equipped        with SPD-20A UV detector and Acclaim SEC-300 4.6 mm×300 mm, 5        μm, 300 Å size exclusion column (11^(st) in series) connected to        Acclaim SEC-1000 4.6 mm×300 mm, 7 μm, 1000 Å (2^(nd) in series)        size exclusion column was assembled. The method used was        isocratic, with 200 mM NaBr, 10 mM Tris pH 8, 1 mM EDTA, and 20%        Acetonitrile as mobile phase and detection at 260 nm. A sample        of polymer conjugate (after RNA trigger addition) was diluted        into mobile phase and injected onto the system. Another sample        of conjugate followed the same dilution scheme but was treated        with 200 mM dithiothreitol for 2 h before injection onto the        system. RNA trigger concentration for both samples was        calculated using an external standard curve. Amount of        conjugated RNA trigger was calculated by subtracting DTT treated        RNA levels from untreated RNA levels. Post-TFF purity of the        conjugate was also determined using this method.

Example 6. RGD Ligands (RGD Mimic)

A. RGD mimic #1-PEG_(n)-HyNic, MW 1272.

-   -   n=4-24 (preferably 8-12) PGP

B. RGD mimic #1a-HyNic, MW 802.8.

C. RGD mimic #1b-HyNic, MW 830.9 (RGD).

Example 7. RGD and PEG Modifying Agents

A. Dipeptide RGD-dipeptide and PEG-dipeptide modifying agents were madeas described in US-2012-0172412-A1 (WO 2012/092373) and US 2015-0045573A1 (WO 2015/021092) (both of which are incorporated herein byreference). FIG. 3-7.

B. RGD-PEG_(n)-FCitFP-TFP and PEG_(n)-FCitFP-TFP modifying agentsynthesis. The modifying agent precursor(di-Boc)RGD(OtBu)-APBA-PEG_(n)-FCitFPro-COOH was prepared using generalFmoc chemistry solid phase synthesis using 2-Cl-Trt resin preloaded withFmoc-Proline-OH. To Resin-Pro-Fmoc was added sequentially (followingFmoc deprotection at each step): FMoc-Phe-OH, Fmoc-Cit-OH, Fmoc-Phe-OH,Fmoc-NH-PEG_(n)-COOH, 4-(N-Fmoc-p-aminophenoxy)-butyric acid (APBA),Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, and diboc-m-guanidino benzoic acid.

(diboc)RGD(OtBu)-APBA-PEG_(n)-FCitFPro-COOH (458 mg, 0.200 mmols) andTFP (66.5 mg, 0.400 mmols) were dissolved in anhydrous DCM (5.0 mL) andcooled to 0° C. in an ice/water bath while stirring under Argon. EDC (77mg, 0.400 mmols) was added and the reaction mixture stirred in anice/water bath at 0° C. for 30 min. Reaction progress was monitored byTLC (8.5:1.5 CHCl₃:MeOH) and was complete after 90 min with no startingmaterial observed by TLC. The reaction mixture was diluted to 100 mLtotal volume with DCM, washed 3×40 mL with DI H₂O (pH=5), and washed1×40 mL aqueous saturated NaCl solution. The organics were then driedover Na₂SO₄, and concentrated on a rotovap to yield 448 mg (92% yield)of a tan/orange foam. The structure was confirmed by ¹H NMR, and ESI MS(Reaction shown above for PEG₂₀ (n=20)).

-   -   (diboc)RGD(OtBu)-APBA-PEG_(n)-FCitFPro-TFP        -   (shown for n=20)

(diboc)RGD(OtBu)-PEG_(n)-FCitFPro-TFP (497 mg, 0.204 mmols) wasdissolved in [9.25:0.75:0.50] TFA:H₂O:Thioanisole (5.0 mL) and stirredat room temperature in a closed flask for 45 min. Reaction completionwas confirmed by MS (ESI, scan neg, 300-3000) with no masses related tostarting material or partially deprotected intermediates observed. Thereaction mixture was then precipitated into 45 mL diethyl ether, spundown, the supernatant poured off, and washed 2×10 mL diethyl ether anddried on high vacuum overnight. The final product was purified on prepHPLC using a Thermo Aquasil C18 5 μm semi prep column, with mobilephases 0.1% TFA in H₂O and ACN. Each injection was 50 mg of crudematerial dissolved in 3.0 mL of 0.1% TFA in [61:39] H₂O:ACN run on agradient of (indicated in % B)39-(5)-39-(35)-43-(5)-95-(10)-95-(2)-39-(5)-39. Each sample forinjection was prepared (dissolved) within 15 minutes of being injectedand positive fractions were pooled in one flask and kept cold in thefreezer until the last injection of the day had finished. The positivefractions were then concentrated on the rotovap with a bath temperatureof 32° C. to dryness, then chased 2× with ACN/Toluene, then 3× with ACNand then dried on high vacuum overnight. Out of 257 mg injected crude,180 mg (70%) was isolated as pure material (Reaction shown above forPEG₂₀ (n=20)).

-   -   RGD-PEG_(n)-FCitFPro-TFP        -   (shown for n=20)

4-(N-Fmoc-p-aminophenoxy)-butyric acid 1 synthesis. p-Nitro-phenol (2)(7.5 g, 53.9 mmole) was combined with ethyl 4-bromobutyrate (8.45 ml, 59mmol) and K₂CO₃ (7.5 g, 54 mmole) in DMF (75 mL). The mixture wasstirred for 2 h at 100° C. DMF was removed and the crude product wasdiluted in a mixture of 3:1 mixture of 2 N NaOH and methanol and stirred4 h at RT. The reaction mixture was acidified with 6 M HCl. The whiteprecipitate was collected to yield 4-(p-Nitrophenyloxy)-butyric acid 3:(10.9 g, 90% yield). 4-(p-Nitrophenyloxy)-butyric acid 3 (37.1 g, 165mmole) was dissolved in MeOH (1 L) with ammonium formate (35 g, 555mmole) and 10% Pd/C (Degussa Type) (3.5 g) was added. The mixture wasrefluxed at 65° C. overnight. The reaction was filtered with celite toyield a reddish brown solid of product 4-(p-Aminophenyloxy)-butyric acid4 (30.5 g, 95% yield).

4-(p-Aminophenyloxy)-butyric acid 4 (5.1 g, 26 mmole) was dissolved in6:4 a mixture of an aqueous saturated NaHCO₃(3.36 g, 40 mmol) in H₂O(450 mL) and THF (300 ml) to make a white slurry. Fmoc-OSu (8.82 g, 26.1mmole) was added and the reaction was stirred for 4 h. The acetone wasremoved, the reaction was acidified (HCl), and the off-white precipitatewas collected and triturated in 1N HCl to yield 9.6 g of product4-(N-Fmoc-p-aminophenoxy)-butyric acid 1 (88% yield).

-   -   diBoc-m-guanidino-benzoic acid 5 was synthesized according to        Riches A G et al. Tetrahedron (2012) 68, p. 9448-9455.

PEGe-FCitFP modifying agents were made using similar chemistry. FIG.1-2.

Example 8. Tetrapeptide Peptide Linkages

A. Tetrapeptide Syntheses. All tetrapeptides were synthesized in thesame manner using standard solid phase Fmoc procedures. Some peptideswere synthesized from commercially available 2-Cl-Trt resin (EMDMillipore, Billerica, Mass.) containing either proline, leucine, oralanine. For other peptides, 2-Cl-Trt resin was loaded with eitherFMOC-PEG_(n)-CO₂H or FMOC-N-methyl-Ala-CO₂H by adding a solution of DMFcontaining the amino acid or PEG (1 eq) and DIEA (2 eq) to 2-Cl-Trtresin for 16 h. Upon completion, resins were capped with MeOH. Stepwiseaddition was performed using PYBOP (4 eq), amino acid (4 eq), and DIEA(8 eq) for coupling and 20% piperdine in DMF for Fmoc de-protection.

After peptide syntheses, the tetrapeptides were reacted with 2 eq ofN-Hydroxysuccinimide (NHS) activated esters of either protectedN-Acetyl-galactosamine, NAG(OAc)₃ (Rozema D B et al. “Protease-triggeredsiRNA Delivery Vehicles.” J Control Release. 2015 Vol. 209:57-66 andU.S. Pat. No. 8,802,773) or PEG_(n) in DMF containing 4 eq DIEA.Following attachment of NAG(OAc)₃ or PEG, the peptides were removed fromresin using HFIP (30%) in DCM for 0.5 h. After solvent removal theresidue was triturated with Et₂O.

Tetrapeptides were either purified and conjugated to activated esters toform modifying agents or conjugated to chromophoreN-(p-Nitrophenyl)ethylenediamine (pNA) without purification to formsubstrates for physiological lability testing. Prior to purification,NAG(OAc)₃-containing substrates were de-acetylated by treatment with TEA(35%) in water (45%) and MeOH (20%) and stirred at room temp. Forpurification, tetrapeptide substrates were separated by HPLC using aThermo Scientific Aquasil C18 reverse-phase column (250×21.2, Waltham,Mass.), eluting a gradient of acetonitrile and water buffered with 0.1%formic acid. Following purification, the substrates were lyophilized.

Attachment of amine-reactive groups to tetrapeptides. 1 eq HPLC purifiedpeptide with N-terminal NAG (RS=NAG(OH)₃ or PEG (R=PEGe) in DMF or DCMwas added to a flame dried flask at 0° C. to give a 0.2 M concentrationof peptide. NHS (3 eq) and N,N′-Dicyclohexylcarbodiimide (DCC) (3 eq)were added and allowed to stir at room temp. under argon overnight toyield the modifying agents. The mixture was partially concentrated,chilled to −20° C., and filtered. All solvents were then removed invacuo. The residue was dissolved in a minimum of DCM and MeOH,precipitated into cold Et₂O and collected by decantation of the solventafter centrifugation. Precipitation into Et₂O was repeated until noresidual DCU (dicyclohexylurea) was detectable. All prepared compoundswere subsequently used without further purification.

Example 9. Polymer Modification

Formation of siRNA delivery conjugate using RGD-PEG-HyNic,RGD-PEG-ACit-PNP, or RDG-PEG-FCitFP-TFP and PEG-dipeptide modifyingagents.

1) Protocol 1. The indicated polymer was reacted with SMPT at a weightratio of 1:0.015 (polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h atRT. The SMPT-modified polymer was then reacted withaldehyde-PEG-dipeptide modifying agent (aldehyde-PEG₁₂-FCit oraldehyde-PEG₂₄-ACit) at desired ratios for 1 h at RT. The modifiedpolymer was then reacted with PEG₁₂-dipeptide modifying agent(PEG₁₂-FCit, PEG₁₂-ACit or PEG₂₄-ACit) at a weight ratio of 1:2(polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modifiedpolymer was then reacted overnight with SATA-RNAi trigger at a weightratio of 1:0.2 (polymer:SATA-RNAi trigger) in 100 mM HEPES, pH 9.0buffer at RT to attach the RNAi trigger. Next, the modified polymer wasreacted with protease cleavable PEG (PEG₁₂-FCit or PEG₁₂-ACit orPEG₂₄-ACit) at a weight ratio of 1:6 (polymer:PEG) in 100 mM HEPES, pH9.0 buffer for 1 h at RT. The resultant conjugate was purified using asephadex G-50 spin column.

-   -   RGD-HyNic (Example 6B) was attached to the modified polymer to        form the full delivery conjugate by reaction with the modified        polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in        50 mM MES, pH 5.0 buffer for a minimum of 4 h at RT. The        conjugate was purified using a sephadex G-50 spin column. RGD        ligand attachment efficiency was determined as described above.

2) Protocol 2. The indicated polymer was reacted with SMPT at a weightratio of 1:0.015 (polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h atRT. The SMPT-modified polymer was then reacted withaldehyde-PEG-dipeptide modifying agent (aldehyde-PEG₂₄-ACit) at a weightratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent(PEG₁₂-FCit, PEG₁₂-ACit or PEG₂₄-ACit) at a weight ratio of 1:2(polymer:PEG) in 100 mM HEPES, pH 9.0 buffer for 1 h at RT. The modifiedpolymer was then reacted overnight with SATA-RNAi trigger at a weightratio of 1:0.2 (polymer:SATA-RNAi trigger) in 100 mM HEPES, pH 9.0buffer at RT to attach the RNAi trigger. Next, the modified polymer wasreacted with protease cleavable-PEG (PEG₁₂-FCit or PEG₁₂-ACit orPEG₂₄-ACit) at a weight ratio of 1:6 (polymer:PEG) in 100 mM HEPES, pH9.0 buffer for 1 h at RT. RGD-HyNic (Example 6) was attached to themodified polymer to form the full conjugate by reaction with themodified polymer at a weight ratio of 1:0.7 (polymer:RGD-HyNic) in 69 mMhydrogen chloride solution (HCl) overnight at RT. RGD ligand attachmentefficiency was determined as described above. 3) Protocol 3. Theindicated polymer was reacted with SMPT at a weight ratio of 1:0.015(polymer:SMPT) in 5 mM HEPES, pH 8.0 buffer for 1 h at RT. TheSMPT-modified polymer was then reacted with aldehyde-PEG-dipeptidemodifying agent (aldehyde-PEG₂₄-ACit) at a weight ratio of 1:0.5(polymer:PEG) and with PEG-dipeptide modifying agent (PEG₁₂-FCit,PEG₁₂-ACit or PEG₂₄-ACit) at a weight ratio of 1:2 (polymer:PEG) in 50mM HEPES, pH 9.0 buffer for 1 h at RT. The modified polymer was thenreacted overnight with SATA-RNAi trigger at a weight ratio of 1:0.2(polymer:SATA-RNAi trigger) in 50 mM HEPES, pH 9.0 buffer at RT toattach the RNAi trigger. Next, the modified polymer was reacted withprotease cleavable-PEG (PEG₁₂-FCit or PEG₁₂-ACit or PEG₂₄-ACit) at aweight ratio of 1:6 (polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 hat RT. RGD-HyNic (Example 6) was attached to the modified polymer toform the full delivery conjugate by reaction with the modified polymerat a weight ratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100 mM MES freeacid solution overnight at RT. RGD targeting ligand conjugationefficiency was determined as described above.

4) Protocol 4. The indicated polymer was reacted with Azido-PEG₄-NHS ata weight ratio of 1:0.015 (polymer:Azido) in 5 mM HEPES, pH 8.0 bufferfor 1 h at RT. The Azido-modified polymer was then reacted withaldehyde-PEG-dipeptide modifying agent (aldehyde-PEG₂₄-ACit) at a weightratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent(PEG₁₂-ACit) at a weight ratio of 1:2 (polymer:PEG) in 50 mM HEPES, pH9.0 buffer for 1 h at RT. The modified polymer was then reactedovernight with Alkyne-RNAi trigger at a weight ratio of 1:0.2(polymer:Alkyne-RNAi trigger) in 50 mM HEPES, pH 9.0 buffer at RT toattach the RNAi trigger. Next, the modified polymer was reacted withprotease cleavable-PEG (PEG₁₂-ACit) at a weight ratio of 1:6(polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. RGD-HyNic(Example 6) was attached to the modified polymer to form the fulldelivery conjugate by reaction with the modified polymer at a weightratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100 mM sodium acetate-aceticacid buffer solution, pH 5.0 overnight at RT. RGD targeting ligandconjugation efficiency was determined as described above.

5) Protocol S. The mono azide-polymer was reacted withaldehyde-PEG-dipeptide modifying agent (aldehyde-PEG₂₄-ACit) at a weightratio of 1:0.5 (polymer:PEG) and with PEG-dipeptide modifying agent(PEG₁₂-ACit) at a weight ratio of 1:2 (polymer:PEG) in 50 mM HEPES, pH9.0 buffer for 1 h at RT. The modified polymer was then reactedovernight with Alkyne-RNAi trigger at a weight ratio of 1:0.2(polymer:Alkyne-RNAi trigger) in 50 mM HEPES, pH 9.0 buffer at RT toattach the RNAi trigger. Next, the modified polymer was reacted withprotease cleavable-PEG (PEG₁₂-ACit) at a weight ratio of 1:6(polymer:PEG) in 50 mM HEPES, pH 9.0 buffer for 1 h at RT. RGD-HyNic(Example 6) was attached to the modified polymer to form the fulldelivery conjugate by reaction with the modified polymer at a weightratio of 1:0.7 (polymer:RGD-HyNic mimic) in 100 mM sodium acetate-aceticacid buffer solution, pH 5.0 overnight at RT. RGD targeting ligandconjugation efficiency was determined as described above.

6) Protocol 6. The mono azide-polymer was reacted with proteasecleavable-RGD agent (RGD-PEG₈-ACit-PNP, RDG-PEG₈-FCitFP-TFP,RGD-PEG₁₅-FCitFP-TFP, RGD-PEG₁₉-FCitFP-TFP, or RGD-PEG₂₀-FCitFP-TFP) atweight ratios of 1:0.125, 1:0.25, 1:0.5, 1:1, 1:1.5, 1:2 (polymer:RGD)in 50 mM HEPES, pH 8.5 buffer for 4 h at RT. The modified polymer wasthen reacted with protease cleavable-PEG agent (PEG₆-ACit-PABC-PNP,PEG₁₂-ACit-PABC-PNP, PEG₁₂-FCit-PABC-PNP, PEG₁₂-FCitFP-TFP) at a weightratio of 1:8 (polymer:PEG) in 50 mM HEPES, pH 8.5 buffer for 2 h at RT.Alkyne-RNAi trigger at a weight ratio of 1:0.3 (polymer:Alkyne-RNAitrigger) was added to the modified polymer in 100 mM sodiumacetate-acetic acid buffer solution, pH 5.0 for 5 days at RT. Thecompleted conjugate was TFF purified and conjugation efficiencydetermined.

7) Protocol 7. The mono azide-polymer was reacted with proteasecleavable-RGD agent (RGD-PEG₂₀-FCitFP-TFP) at weight ratio of 1:1(polymer:RGD) in 50 mM HEPES, pH 8.5 buffer for 2 h at RT. The modifiedpolymer was then reacted with protease cleavable-PEG agent(PEG₁₂-ACit-PABC-PNP) at a weight ratio of 1:8 (polymer:PEG) in 50 mMHEPES, pH 8.5 buffer for 2 h at RT. The modified polymer was then TFFpurified. Alkyne-RNAi trigger at a weight ratio of 1:0.4(polymer:Alkyne-RNAi trigger) was added to the TFF purified polymer for3 days at 37° C.

Example 10. In Vitro Analysis of Hf2α RNAi Triggers

Candidate sequences were identified by in silico analysis and screenedas chemically modified canonical siRNAs in vitro. For screeningpurposes, the human EPAS1 (Hif2a) cDNA sequence (accession #NM_001430)was synthesized and cloned (DNA 2.0, Menlo Park, Calif.) into acommercially-available reporter-based screening plasmid, psiCHECK2(Promega, Madison, Wis.) which generated a Renilla luciferase/EPAS1fusion mRNA. For RNAi trigger efficacy evaluation, Hep3B cells, a humanhepatocellular carcinoma line, were plated at ˜10,000 cells per well in96-well format. Each of the 187 EPAS1 RNAi triggers, in two subsets, wasco-transfected at two concentrations, 1 nM and 0.1 nM, with 25 ngEPAS1-psiCHECK2 plasmid DNA per well and 0.2 μL LipoFectamine 2000 (LifeTechnologies) per well. Gene knockdown was determined by measuringRenilla luciferase levels normalized to the levels ofconstitutively-expressed firefly luciferase, also present on thepsiCHECK-2 plasmid, using the Dual Luciferase Reporter Assay (Promega,Madison, Wis.) Table 5.

TABLE 5A Unmodified Hif2α RNAi trigger antisense strand and sense strand sequences. SEQ SEQ ID Sense Strand IDAntisense Strand NO. Sequence (5′ → 3′) NO. Sequence (5′ → 3′) 285GAGACUGUAUGGUCAGCUC 478 GAGCUGACCAUACAGUCUC 286 CUCCGACUCCUUCCGACUC 479GAGUCGGAAGGAGUCGGAG 287 UCCGACUCCCAGCAUUCGA 480 UCGAAUGCUGGGAGUCGGA 288CGACUCCCAGCAUUCGAGC 481 GCUCGAAUGCUGGGAGUCG 289 GACUCCCAGCAUUCGAGCC 482GGCUCGAAUGCUGGGAGUC 290 CAGGUGCUCGGCGUCUGAA 483 UUCAGACGCCGAGCACCUG 291GUGCUCGGCGUCUGAACGU 484 ACGUUCAGACGCCGAGCAC 292 UCGGCGUCUGAACGUCUCA 485UGAGACGUUCAGACGCCGA 293 GGCGUCUGAACGUCUCAAA 486 UUUGAGACGUUCAGACGCC 294CGUCUGAACGUCUCAAAGG 487 CCUUUGAGACGUUCAGACG 295 AAAAGGAGUAGCUCGGAGA 488UCUCCGAGCUACUCCUUUU 296 GGGUUUCAUUGCCGUGGUG 489 CACCACGGCAAUGAAACCC 297UUCAUGGGACUUACACAGG 490 CCUGUGUAAGUCCCAUGAA 298 GGGACUUACACAGGUGGAG 491CUCCACCUGUGUAAGUCCC 299 ACACAGGUGGAGCUAACAG 492 CUGUUAGCUCCACCUGUGU 300GAGCUAACAGGACAUAGUA 493 UACUAUGUCCUGUUAGCUC 301 GCUAACAGGACAUAGUAUC 494GAUACUAUGUCCUGUUAGC 302 CUAACAGGACAUAGUAUCU 495 AGAUACUAUGUCCUGUUAG 303GGACAUAGUAUCUUUGACU 496 AGUCAAAGAUACUAUGUCC 304 UCUUUGACUUCACUCAUCC 497GGAUGAGUGAAGUCAAAGA 305 UCACUCAUCCCUGCGACCA 498 UGGUCGCAGGGAUGAGUGA 306GAGAUUCGUGAGAACCUGA 499 UCAGGUUCUCACGAAUCUC 307 UUCGUGAGAACCUGAGUCU 500AGACUCAGGUUCUCACGAA 308 UCGUGAGAACCUGAGUCUC 501 GAGACUCAGGUUCUCACGA 309GACAUGUCCACAGAGCGGG 502 CCCGCUCUGUGGACAUGUC 310 GCGGGACUUCUUCAUGAGG 503CCUCAUGAAGAAGUCCCGC 311 GGAUGAAGUGCACGGUCAC 504 GUGACCGUGCACUUCAUCC 312CACGGUCACCAACAGAGGC 505 GCCUCUGUUGGUGACCGUG 313 UCACCAACAGAGGCCGUAC 506GUACGGCCUCUGUUGGUGA 314 CACCAACAGAGGCCGUACU 507 AGUACGGCCUCUGUUGGUG 315AGGCCGUACUGUCAACCUC 508 GAGGUUGACAGUACGGCCU 316 UCCUCACAAUAGUCUGUGU 509ACACAGACUAUUGUGAGGA 317 AAUAGUCUGUGUGGCUACA 510 UGUAGCCACACAGACUAUU 318CAGAACUGAUUGGUUACCA 511 UGGUAACCAAUCAGUUCUG 319 AGAACUGAUUGGUUACCAC 512GUGGUAACCAAUCAGUUCU 320 CUGAUUGGUUACCACCCUG 513 CAGGGUGGUAACCAAUCAG 321UUGGCCGCUCAGCCUAUGA 514 UCAUAGGCUGAGCGGCCAA 322 UAUGAAUUCUACCAUGCGC 515GCGCAUGGUAGAAUUCAUA 323 AUGAAUUCUACCAUGCGCU 516 AGCGCAUGGUAGAAUUCAU 324UGAAUUCUACCAUGCGCUA 517 UAGCGCAUGGUAGAAUUCA 325 GAAUUCUACCAUGCGCUAG 518CUAGCGCAUGGUAGAAUUC 326 AAUUCUACCAUGCGCUAGA 519 UCUAGCGCAUGGUAGAAUU 327UCUACCAUGCGCUAGACUC 520 GAGUCUAGCGCAUGGUAGA 328 AUGCGCUAGACUCCGAGAA 521UUCUCGGAGUCUAGCGCAU 329 UGCGCUAGACUCCGAGAAC 522 GUUCUCGGAGUCUAGCGCA 330GUAAGUGGCCAGUACCGGA 523 UCCGGUACUGGCCACUUAC 331 UAAGUGGCCAGUACCGGAU 524AUCCGGUACUGGCCACUUA 332 CCAGUACCGGAUGCUCGCA 525 UGCGAGCAUCCGGUACUGG 333AGUACCGGAUGCUCGCAAA 526 UUUGCGAGCAUCCGGUACU 334 UACCGGAUGCUCGCAAAGC 527GCUUUGCGAGCAUCCGGUA 335 UGCUCGCAAAGCAUGGGGG 528 CCCCCAUGCUUUGCGAGCA 336CGCAAAGCAUGGGGGCUAC 529 GUAGCCCCCAUGCUUUGCG 337 AGCAUGGGGGCUACGUGUG 530CACACGUAGCCCCCAUGCU 338 GCAUGGGGGCUACGUGUGG 531 CCACACGUAGCCCCCAUGC 339CAUCUACAACCCUCGCAAC 532 GUUGCGAGGGUUGUAGAUG 340 AUCUACAACCCUCGCAACC 533GGUUGCGAGGGUUGUAGAU 341 CUACAACCCUCGCAACCUG 534 CAGGUUGCGAGGGUUGUAG 342UACAACCCUCGCAACCUGC 535 GCAGGUUGCGAGGGUUGUA 343 UUUGAUAGCAGUGGCAAGG 536CCUUGCCACUGCUAUCAAA 344 AGUAACUUCCUAUUCACCA 537 UGGUGAAUAGGAAGUUACU 345UCGGGAAUCAGAACUUCGA 538 UCGAAGUUCUGAUUCCCGA 346 CUGCUCCACGCCCAAUAGC 539GCUAUUGGGCGUGGAGCAG 347 UGCUCCACGCCCAAUAGCC 540 GGCUAUUGGGCGUGGAGCA 348GCUCCACGCCCAAUAGCCC 541 GGGCUAUUGGGCGUGGAGC 349 ACGCCCAAUAGCCCUGAAG 542CUUCAGGGCUAUUGGGCGU 350 CAUCUUUGGAUAACGACCU 543 AGGUCGUUAUCCAAAGAUG 351CAAUGCAGUACCCAGACGG 544 CCGUCUGGGUACUGCAUUG 352 AUGCAGUACCCAGACGGAU 545AUCCGUCUGGGUACUGCAU 353 AGUACCCAGACGGAUUUCA 546 UGAAAUCCGUCUGGGUACU 354CUGUAGCCCCGCACAGUCC 547 GGACUGUGCGGGGCUACAG 355 AUCUUCUUUGAUGCCGGAA 548UUCCGGCAUCAAAGAAGAU 356 CUUUGAUGCCGGAAGCAAA 549 UUUGCUUCCGGCAUCAAAG 357GAUGCCGGAAGCAAAGCAU 550 AUGCUUUGCUUCCGGCAUC 358 AUGCCGGAAGCAAAGCAUC 551GAUGCUUUGCUUCCGGCAU 359 GCCGGAAGCAAAGCAUCCC 552 GGGAUGCUUUGCUUCCGGC 360CCCCCAGAUCCACCAUUAC 553 GUAAUGGUGGAUCUGGGGG 361 AGAUCCACCAUUACAUUUU 554AAAAUGUAAUGGUGGAUCU 362 AUUUUGGGCCCACAAAGUG 555 CACUUUGUGGGCCCAAAAU 363UUUUGGGCCCACAAAGUGG 556 CCACUUUGUGGGCCCAAAA 364 UUUGGGCCCACAAAGUGGG 557CCCACUUUGUGGGCCCAAA 365 CCACAAAGUGGGCCGUCGG 558 CCGACGGCCCACUUUGUGG 366CACAAAGUGGGCCGUCGGG 559 CCCGACGGCCCACUUUGUG 367 AGUGGGCCGUCGGGGAUCA 560UGAUCCCCGACGGCCCACU 368 AAAGGGUUUUGGGGCUCGA 561 UCGAGCCCCAAAACCCUUU 369GGCUCGAGGCCCAGACGUG 562 CACGUCUGGGCCUCGAGCC 370 GCUCGAGGCCCAGACGUGC 563GCACGUCUGGGCCUCGAGC 371 CUCGAGGCCCAGACGUGCU 564 AGCACGUCUGGGCCUCGAG 372GGUAGCCCUCUCCAACAAG 565 CUUGUUGGAGAGGGCUACC 373 CUUUGAUGCCGGACAAGCC 566GGCUUGUCCGGCAUCAAAG 374 UUUGAUGCCGGACAAGCCA 567 UGGCUUGUCCGGCAUCAAA 375UUGAUGCCGGACAAGCCAC 568 GUGGCUUGUCCGGCAUCAA 376 GGACAAGCCACUGAGCGCA 569UGCGCUCAGUGGCUUGUCC 377 ACAAGCCACUGAGCGCAAA 570 UUUGCGCUCAGUGGCUUGU 378GGACUACAGCCUGUCGUCA 571 UGACGACAGGCUGUAGUCC 379 GACUACAGCCUGUCGUCAG 572CUGACGACAGGCUGUAGUC 380 CUACAGCCUGUCGUCAGCC 573 GGCUGACGACAGGCUGUAG 381CCUGUCGUCAGCCCACAAG 574 CUUGUGGGCUGACGACAGG 382 GCAUGGCAAGCCGGCUGCU 575AGCAGCCGGCUUGCCAUGC 383 CUGACCAGAUAUGACUGUG 576 CACAGUCAUAUCUGGUCAG 384GAUAUGACUGUGAGGUGAA 577 UUCACCUCACAGUCAUAUC 385 GGUGAACGUGCCCGUGCUG 578CAGCACGGGCACGUUCACC 386 UACAAGAUGGACUUACCUG 579 CAGGUAAGUCCAUCUUGUA 387GGACUUACCUGGCAGACUU 580 AAGUCUGCCAGGUAAGUCC 388 UUUUUCUGAGAUGCUCACU 581AGUGAGCAUCUCAGAAAAA 389 AGUACACAAUUGUUUUACC 582 GGUAAAACAAUUGUGUACU 390ACAAGUUUGGUGCAUGUCU 583 AGACAUGCACCAAACUUGU 391 ACUAAAAAGAUUCCUCGUU 584AACGAGGAAUCUUUUUAGU 392 AGGGUCAACUCCAACGUAU 585 AUACGUUGGAGUUGACCCU 393GGGUCAACUCCAACGUAUG 586 CAUACGUUGGAGUUGACCC 394 GUCAACUCCAACGUAUGUG 587CACAUACGUUGGAGUUGAC 395 UCAACUCCAACGUAUGUGG 588 CCACAUACGUUGGAGUUGA 396CAACUCCAACGUAUGUGGU 589 ACCACAUACGUUGGAGUUG 397 CUCCAACGUAUGUGGUUAU 590AUAACCACAUACGUUGGAG 398 UCCAACGUAUGUGGUUAUC 591 GAUAACCACAUACGUUGGA 399CCAACGUAUGUGGUUAUCU 592 AGAUAACCACAUACGUUGG 400 AACGUAUGUGGUUAUCUGU 593ACAGAUAACCACAUACGUU 401 UUAUAUCUGGGUUAAGUGU 594 ACACUUAACCCAGAUAUAA 402CCACGGCCUGUACGGACAC 595 GUGUCCGUACAGGCCGUGG 403 ACGGCCUGUACGGACACUG 596CAGUGUCCGUACAGGCCGU 404 UGUCGGCUUUUUGCCAUCU 597 AGAUGGCAAAAAGCCGACA 405GUCGGCUUUUUGCCAUCUG 598 CAGAUGGCAAAAAGCCGAC 406 AUCUGUGAUAUGCCAUAGG 599CCUAUGGCAUAUCACAGAU 407 UGCCAUAGGUGUGACAAUC 600 GAUUGUCACACCUAUGGCA 408CCAUAGGUGUGACAAUCCG 601 CGGAUUGUCACACCUAUGG 409 CAUAGGUGUGACAAUCCGA 602UCGGAUUGUCACACCUAUG 410 AUAGGUGUGACAAUCCGAG 603 CUCGGAUUGUCACACCUAU 411GGUGUGACAAUCCGAGCAG 604 CUGCUCGGAUUGUCACACC 412 ACAAUCCGAGCAGUGGAGU 605ACUCCACUGCUCGGAUUGU 413 CCGAGCAGUGGAGUCAUUC 606 GAAUGACUCCACUGCUCGG 414GGGAGCACUGCGCGCUAUC 607 GAUAGCGCGCAGUGCUCCC 415 GGAGCACUGCGCGCUAUCC 608GGAUAGCGCGCAGUGCUCC 416 AGCACUGCGCGCUAUCCCC 609 GGGGAUAGCGCGCAGUGCU 417UAUUGCUGCCAAGAGGGUC 610 GACCCUCUUGGCAGCAAUA 418 GGUCUGAUGGCACGUUGUG 611CACAACGUGCCAUCAGACC 419 CUGAUGGCACGUUGUGGGG 612 CCCCACAACGUGCCAUCAG 420GGCACGUUGUGGGGUCGGG 613 CCCGACCCCACAACGUGCC 421 GCACGUUGUGGGGUCGGGG 614CCCCGACCCCACAACGUGC 422 CACGUUGUGGGGUCGGGGG 615 CCCCCGACCCCACAACGUG 423GCGGGGAAGUGCUCUAACU 616 AGUUAGAGCACUUCCCCGC 424 CGGGGAAGUGCUCUAACUU 617AAGUUAGAGCACUUCCCCG 425 UUAAGGUUUUGUUGCUAGC 618 GCUAGCAACAAAACCUUAA 426GUUGCUAGCCCUUCAAGUG 619 CACUUGAAGGGCUAGCAAC 427 GAGCUAUGUGACUCGGAUG 620CAUCCGAGUCACAUAGCUC 428 GCUAUGUGACUCGGAUGGU 621 ACCAUCCGAGUCACAUAGC 429CGGAUGGUCUUUCACACGG 622 CCGUGUGAAAGACCAUCCG 430 GAUGGUCUUUCACACGGCA 623UGCCGUGUGAAAGACCAUC 431 UGGUCUUUCACACGGCACA 624 UGUGCCGUGUGAAAGACCA 432AACUACCAUGAGAUGGUUU 625 AAACCAUCUCAUGGUAGUU 433 UACCAUGAGAUGGUUUAGA 626UCUAAACCAUCUCAUGGUA 434 CCAAGCUCACGACCUUGGA 627 UCCAAGGUCGUGAGCUUGG 435ACGACCUUGGAGCCCCGUG 628 CACGGGGCUCCAAGGUCGU 436 GGGUAAGAGGGACGACACC 629GGUGUCGUCCCUCUUACCC 437 GGUAAGAGGGACGACACCU 630 AGGUGUCGUCCCUCUUACC 438GUAAGAGGGACGACACCUC 631 GAGGUGUCGUCCCUCUUAC 439 UGGUUUUUCAAUACCAAUU 632AAUUGGUAUUGAAAAACCA 440 UUCAAUACCAAUUACAUGG 633 CCAUGUAAUUGGUAUUGAA 441AUACCAAUUACAUGGAACU 634 AGUUCCAUGUAAUUGGUAU 442 CCAACUAUUUAGUAAGCCC 635GGGCUUACUAAAUAGUUGG 443 AACUAUUUAGUAAGCCCGG 636 CCGGGCUUACUAAAUAGUU 444ACUAUUUAGUAAGCCCGGA 637 UCCGGGCUUACUAAAUAGU 445 AGAAAUUCCUUAGUCAUGG 638CCAUGACUAAGGAAUUUCU 446 CAUUAAGGGCAUUUUACCC 639 GGGUAAAAUGCCCUUAAUG 447UAAGGGCAUUUUACCCUUG 640 CAAGGGUAAAAUGCCCUUA 448 AGCUUCAUAUUAACCCUAC 641GUAGGGUUAAUAUGAAGCU 449 UAUUAACCCUACCUGUCAA 642 UUGACAGGUAGGGUUAAUA 450UUAACCCUACCUGUCAACG 643 CGUUGACAGGUAGGGUUAA 451 ACCCUACCUGUCAACGUAA 644UUACGUUGACAGGUAGGGU 452 CCCUACCUGUCAACGUAAC 645 GUUACGUUGACAGGUAGGG 453CCUACCUGUCAACGUAACG 646 CGUUACGUUGACAGGUAGG 454 CUACCUGUCAACGUAACGA 647UCGUUACGUUGACAGGUAG 455 UACCUGUCAACGUAACGAU 648 AUCGUUACGUUGACAGGUA 456ACCUGUCAACGUAACGAUU 649 AAUCGUUACGUUGACAGGU 457 CCUGUCAACGUAACGAUUU 650AAAUCGUUACGUUGACAGG 458 CUGUCAACGUAACGAUUUC 651 GAAAUCGUUACGUUGACAG 459UGUCAACGUAACGAUUUCA 652 UGAAAUCGUUACGUUGACA 460 UCAACGUAACGAUUUCAUG 653CAUGAAAUCGUUACGUUGA 461 ACGUAACGAUUUCAUGAAC 654 GUUCAUGAAAUCGUUACGU 462UAUUAUAUUGUCGAAUUCC 655 GGAAUUCGACAAUAUAAUA 463 UUAUAUUGUCGAAUUCCUA 656UAGGAAUUCGACAAUAUAA 464 UAUUGUCGAAUUCCUACUG 657 CAGUAGGAAUUCGACAAUA 465GAAUUCCUACUGACAACAU 658 AUGUUGUCAGUAGGAAUUC 466 UCCUACUGACAACAUUAUA 659UAUAAUGUUGUCAGUAGGA 467 UAUAACUGUAUGGGAGCUU 660 AAGCUCCCAUACAGUUAUA 468UAACUGUAUGGGAGCUUAA 661 UUAAGCUCCCAUACAGUUA 469 UGUAUGGGAGCUUAACUUU 662AAAGUUAAGCUCCCAUACA 470 UUGACACUGGUAUCUUAUU 663 AAUAAGAUACCAGUGUCAA 471AAGUAUUCUGAUCCUACCA 664 UGGUAGGAUCAGAAUACUU 472 CAACGUAACGAUUUCAUGA 665UUCAUGAAAUCGUUACGUU AA GGC 473 UAUAUCAACGUAACGAUUU 666UUCAUGAAAUCGUUACGUU CAUGAAA GGCU 474 UAUAUCAACGUAACGAUUU 667UUCAUGAAAUCGUUACGUU CAUGAAA GGCUAU 475 UAUAUCAACGUAACGAUUU 668UUCAUGAAAUCGUUACGUU CAUGAAA GGCUGU 476 UAUAUCGACGUAACGAUUU 669UUCAUGAAAUCGUUACGUC CAUGAAA GGCUAU 477 UAUACGUAACGAUUUCAUG 670UUCAUGAAAUCGUUACGU AAA

TABLE 5B Hif2α RNAi trigger sequences having modified nucleotides.Duplex SEQ SEQ ID ID Sense Strand Sequence ID Antisense Strand SequenceNo. No. (5′ → 3′) No. (5′ → 3′) 2231 671 GfaGfaCfuGfuAfuGfgUfcAfgCfuAfdT 858 dTAfgCfuGfaCfcAfuAfcAfgUfcUfcdTsdT 2232 672CfuCfcGfaCfuCfcUfuCfcGfaCfuAfdT  859 dTAfgUfcGfgAfaGfgAfgUfcGfgAfgdTsdT2233 673 UfcCfgAfcUfcCfcAfgCfaUfuCfgAfdT  860dTCfgAfaUfgCfuGfgGfaGfuCfgGfadTsdT 2234 674CfgAfcUfcCfcAfgCfaUfuCfgAfgAfdT  861 dTCfuCfgAfaUfgCfuGfgGfaGfuCfgdTsdT2235 675 GfaCfuCfcCfaGfcAfuUfcGfaGfcAfdT  862dTGfcUfcGfaAfuGfcUfgGfgAfgUfcdTsdT 2236 676CfaGfgUfgCfuCfgGfcGfuCfuGfaAfdT  863 dTUfcAfgAfcGfcCfgAfgCfaCfcUfgdTsdT2237 677 GfuGfcUfcGfgCfgUfcUfgAfaCfgAfdT  864dTCfgUfuCfaGfaCfgCfcGfaGfcAfcdTsdT 2238 678UfcGfgCfgUfcUfgAfaCfgUfcUfcAfdT  865 dTGfaGfaCfgUfuCfaGfaCfgCfcGfadTsdT2239 679 GfgCfgUfcUfgAfaCfgUfcUfcAfaAfdT  866dTUfuGfaGfaCfgUfuCfaGfaCfgCfcdTsdT 2240 680CfgUfcUfgAfaCfgUfcUfcAfaAfgAfdT  867 dTCfuUfuGfaGfaCfgUfuCfaGfaCfgdTsdT2241 681 AfaAfaGfgAfgUfaGfcUfcGfgAfgAfdT  868dTCfuCfcGfaGfcUfaCfuCfcUfuUfudTsdT 2242 682GfgGfuUfuCfaUfuGfcCfgUfgGfuAfdT  869 dTAfcCfaCfgGfcAfaUfgAfaAfcCfcdTsdT2243 683 UfuCfaUfgGfgAfcUfuAfcAfcAfgAfdT  870dTCfuGfuGfuAfaGfuCfcCfaUfgAfadTsdT 2244 684GfgGfaCfuUfaCfaCfaGfgUfgGfaAfdT  871 dTUfcCfaCfcUfgUfgUfaAfgUfcCfcdTsdT2245 685 AfcAfcAfgGfuGfgAfgCfuAfaCfaAfdT  872dTUfgUfuAfgCfuCfcAfcCfuGfuGfudTsdT 2246 686GfaGfcUfaAfcAfgGfaCfaUfaGfuAfdT  873 dTAfcUfaUfgUfcCfuGfuUfaGfcUfcdTsdT2247 687 GfcUfaAfcAfgGfaCfaUfaGfuAfuAfdT  874dTAfuAfcUfaUfgUfcCfuGfuUfaGfcdTsdT 2248 688CfuAfaCfaGfgAfcAfuAfgUfaUfcAfdT  875 dTGfaUfaCfuAfuGfuCfcUfgUfuAfgdTsdT2249 689 GfgAfcAfuAfgUfaUfcUfuUfgAfcAfdT  876dTGfuCfaAfaGfaUfaCfuAfuGfuCfcdTsdT 2250 690UfcUfuUfgAfcUfuCfaCfuCfaUfcAfdT  877 dTGfaUfgAfgUfgAfaGfuCfaAfaGfadTsdT2251 691 UfcAfcUfcAfuCfcCfuGfcGfaCfcAfdT  878dTGfgUfcGfcAfgGfgAfuGfaGfuGfadTsdT 2252 692GfaGfaUfuCfgUfgAfgAfaCfcUfgAfdT  879 dTCfaGfgUfuCfuCfaCfgAfaUfcUfcdTsdT2253 693 UfuCfgUfgAfgAfaCfcUfgAfgUfcAfdT  880dTGfaCfuCfaGfgUfuCfuCfaCfgAfadTsdT 2254 694UfcGfuGfaGfaAfcCfuGfaGfuCfuAfdT  881 dTAfgAfcUfcAfgGfuUfcUfcAfcGfadTsdT2255 695 GfaCfaUfgUfcCfaCfaGfaGfcGfgAfdT  882dTCfcGfcUfcUfgUfgGfaCfaUfgUfcdTsdT 2256 696GfcGfgGfaCfuUfcUfuCfaUfgAfgAfdT  883 dTCfuCfaUfgAfaGfaAfgUfcCfcGfcdTsdT2257 697 GfgAfuGfaAfgUfgCfaCfgGfuCfaAfdT  884dTUfgAfcCfgUfgCfaCfuUfcAfuCfcdTsdT 2258 698CfaCfgGfuCfaCfcAfaCfaGfaGfgAfdT  885 dTCfcUfcUfgUfuGfgUfgAfcCfgUfgdTsdT2259 699 UfcAfcCfaAfcAfgAfgGfcCfgUfaAfdT  886dTUfaCfgGfcCfuCfuGfuUfgGfuGfadTsdT 2260 700CfaCfcAfaCfaGfaGfgCfcGfuAfcAfdT  887 dTGfuAfcGfgCfcUfcUfgUfuGfgUfgdTsdT2261 701 AfgGfcCfgUfaCfuGfuCfaAfcCfuAfdT  888dTAfgGfuUfgAfcAfgUfaCfgGfcCfudTsdT 2262 702UfcCfuCfaCfaAfuAfgUfcUfgUfgAfdT  889 dTCfaCfaGfaCfuAfuUfgUfgAfgGfadTsdT2263 703 AfaUfaGfuCfuGfuGfuGfgCfuAfcAfdT  890dTGfuAfgCfcAfcAfcAfgAfcUfaUfudTsdT 2264 704CfaGfaAfcUfgAfuUfgGfuUfaCfcAfdT  891 dTGfgUfaAfcCfaAfuCfaGfuUfcUfgdTsdT2265 705 AfgAfaCfuGfaUfuGfgUfuAfcCfaAfdT  892dTUfgGfuAfaCfcAfaUfcAfgUfuCfudTsdT 2266 706CfuGfaUfuGfgUfuAfcCfaCfcCfuAfdT  893 dTAfgGfgUfgGfuAfaCfcAfaUfcAfgdTsdT2267 707 UfuGfgCfcGfcUfcAfgCfcUfaUfgAfdT  894dTCfaUfaGfgCfuGfaGfcGfgCfcAfadTsdT 2268 708UfaUfgAfaUfuCfuAfcCfaUfgCfgAfdT  895 dTCfgCfaUfgGfuAfgAfaUfuCfaUfadTsdT2269 709 AfuGfaAfuUfcUfaCfcAfuGfcGfcAfdT  896dTGfcGfcAfuGfgUfaGfaAfuUfcAfudTsdT 2270 710UfgAfaUfuCfuAfcCfaUfgCfgCfuAfdT  897 dTAfgCfgCfaUfgGfuAfgAfaUfuCfadTsdT2271 711 GfaAfuUfcUfaCfcAfuGfcGfcUfaAfdT  898dTUfaGfcGfcAfuGfgUfaGfaAfuUfcdTsdT 2272 712AfaUfuCfuAfcCfaUfgCfgCfuAfgAfdT  899 dTCfuAfgCfgCfaUfgGfuAfgAfaUfudTsdT2273 713 UfcUfaCfcAfuGfcGfcUfaGfaCfuAfdT  900dTAfgUfcUfaGfcGfcAfuGfgUfaGfadTsdT 2274 714AfuGfcGfcUfaGfaCfuCfcGfaGfaAfdT  901 dTUfcUfcGfgAfgUfcUfaGfcGfcAfudTsdT2275 715 UfgCfgCfuAfgAfcUfcCfgAfgAfaAfdT  902dTUfuCfuCfgGfaGfuCfuAfgCfgCfadTsdT 2276 716GfuAfaGfuGfgCfcAfgUfaCfcGfgAfdT  903 dTCfcGfgUfaCfuGfgCfcAfcUfuAfcdTsdT2277 717 UfaAfgUfgGfcCfaGfuAfcCfgGfaAfdT  904dTUfcCfgGfuAfcUfgGfcCfaCfuUfadTsdT 2278 718CfcAfgUfaCfcGfgAfuGfcUfcGfcAfdT  905 dTGfcGfaGfcAfuCfcGfgUfaCfuGfgdTsdT2279 719 AfgUfaCfcGfgAfuGfcUfcGfcAfaAfdT  906dTUfuGfcGfaGfcAfuCfcGfgUfaCfudTsdT 2280 720UfaCfcGfgAfuGfcUfcGfcAfaAfgAfdT  907 dTCfuUfuGfcGfaGfcAfuCfcGfgUfadTsdT2281 721 UfgCfuCfgCfaAfaGfcAfuGfgGfgAfdT  908dTCfcCfcAfuGfcUfuUfgCfgAfgCfadTsdT 2282 722CfgCfaAfaGfcAfuGfgGfgGfcUfaAfdT  909 dTUfaGfcCfcCfcAfuGfcUfuUfgCfgdTsdT2283 723 AfgCfaUfgGfgGfgCfuAfcGfuGfuAfdT  910dTAfcAfcGfuAfgCfcCfcCfaUfgCfudTsdT 2284 724GfcAfuGfgGfgGfcUfaCfgUfgUfgAfdT  911 dTCfaCfaCfgUfaGfcCfcCfcAfuGfcdTsdT2285 725 CfaUfcUfaCfaAfcCfcUfcGfcAfaAfdT  912dTUfuGfcGfaGfgGfuUfgUfaGfaUfgdTsdT 2286 726AfuCfuAfcAfaCfcCfuCfgCfaAfcAfdT  913 dTGfuUfgCfgAfgGfgUfuGfuAfgAfudTsdT2287 727 CfuAfcAfaCfcCfuCfgCfaAfcCfuAfdT  914dTAfgGfuUfgCfgAfgGfgUfuGfuAfgdTsdT 2288 728UfaCfaAfcCfcUfcGfcAfaCfcUfgAfdT  915 dTCfaGfgUfuGfcGfaGfgGfuUfgUfadTsdT2289 729 UfuUfgAfuAfgCfaGfuGfgCfaAfgAfdT  916dTCfuUfgCfcAfcUfgCfuAfuCfaAfadTsdT 2290 730AfgUfaAfcUfuCfcUfaUfuCfaCfcAfdT  917 dTGfgUfgAfaUfaGfgAfaGfuUfaCfudTsdT2291 731 UfcGfgGfaAfuCfaGfaAfcUfuCfgAfdT  918dTCfgAfaGfuUfcUfgAfuUfcCfcGfadTsdT 2292 732CfuGfcUfcCfaCfgCfcCfaAfuAfgAfdT  919 dTCfuAfuUfgGfgCfgUfgGfaGfcAfgdTsdT2293 733 UfgCfuCfcAfcGfcCfcAfaUfaGfcAfdT  920dTGfcUfaUfuGfgGfcGfuGfgAfgCfadTsdT 2294 734GfcUfcCfaCfgCfcCfaAfuAfgCfcAfdT  921 dTGfgCfuAfuUfgGfgCfgUfgGfaGfcdTsdT2295 735 AfcGfcCfcAfaUfaGfcCfcUfgAfaAfdT  922dTUfuCfaGfgGfcUfaUfuGfgGfcGfudTsdT 2296 736CfaUfcUfuUfgGfaUfaAfcGfaCfcAfdT  923 dTGfgUfcGfuUfaUfcCfaAfaGfaUfgdTsdT2297 737 CfaAfuGfcAfgUfaCfcCfaGfaCfgAfdT  924dTCfgUfcUfgGfgUfaCfuGfcAfuUfgdTsdT 2298 738AfuGfcAfgUfaCfcCfaGfaCfgGfaAfdT  925 dTUfcCfgUfcUfgGfgUfaCfuGfcAfudTsdT2299 739 AfgUfaCfcCfaGfaCfgGfaUfuUfcAfdT  926dTGfaAfaUfcCfgUfcUfgGfgUfaCfudTsdT 2300 740CfuGfuAfgCfcCfcGfcAfcAfgUfcAfdT  927 dTGfaCfuGfuGfcGfgGfgCfuAfcAfgdTsdT2301 741 AfuCfuUfcUfuUfgAfuGfcCfgGfaAfdT  928dTUfcCfgGfcAfuCfaAfaGfaAfgAfudTsdT 2302 742CfuUfuGfaUfgCfcGfgAfaGfcAfaAfdT  929 dTUfuGfcUfuCfcGfgCfaUfcAfaAfgdTsdT2303 743 GfaUfgCfcGfgAfaGfcAfaAfgCfaAfdT  930dTUfgCfuUfuGfcUfuCfcGfgCfaUfcdTsdT 2304 744AfuGfcCfgGfaAfgCfaAfaGfcAfuAfdT  931 dTAfuGfcUfuUfgCfuUfcCfgGfcAfudTsdT2305 745 GfcCfgGfaAfgCfaAfaGfcAfuCfcAfdT  932dTGfgAfuGfcUfuUfgCfuUfcCfgGfcdTsdT 2306 746CfcCfcCfaGfaUfcCfaCfcAfuUfaAfdT  933 dTUfaAfuGfgUfgGfaUfcUfgGfgGfgdTsdT2307 747 AfgAfuCfcAfcCfaUfuAfcAfuUfuAfdT  934dTAfaAfuGfuAfaUfgGfuGfgAfuCfudTsdT 2308 748AfuUfuUfgGfgCfcCfaCfaAfaGfuAfdT  935 dTAfcUfuUfgUfgGfgCfcCfaAfaAfudTsdT2309 749 UfuUfuGfgGfcCfcAfcAfaAfgUfgAfdT  936dTCfaCfuUfuGfuGfgGfcCfcAfaAfadTsdT 2310 750UfuUfgGfgCfcCfaCfaAfaGfuGfgAfdT  937 dTCfcAfcUfuUfgUfgGfgCfcCfaAfadTsdT2311 751 CfcAfcAfaAfgUfgGfgCfcGfuCfgAfdT  938dTCfgAfcGfgCfcCfaCfuUfuGfuGfgdTsdT 2312 752CfaCfaAfaGfuGfgGfcCfgUfcGfgAfdT  939 dTCfcGfaCfgGfcCfcAfcUfuUfgUfgdTsdT2313 753 AfgUfgGfgCfcGfuCfgGfgGfaUfcAfdT  940dTGfaUfcCfcCfgAfcGfgCfcCfaCfudTsdT 2314 754AfaAfgGfgUfuUfuGfgGfgCfuCfgAfdT  941 dTCfgAfgCfcCfcAfaAfaCfcCfuUfudTsdT2315 755 GfgCfuCfgAfgGfcCfcAfgAfcGfuAfdT  942dTAfcGfuCfuGfgGfcCfuCfgAfgCfcdTsdT 2316 756GfcUfcGfaGfgCfcCfaGfaCfgUfgAfdT  943 dTCfaCfgUfcUfgGfgCfcUfcGfaGfcdTsdT2317 757 CfuCfgAfgGfcCfcAfgAfcGfuGfcAfdT  944dTGfcAfcGfuCfuGfgGfcCfuCfgAfgdTsdT 2318 758GfgUfaGfcCfcUfcUfcCfaAfcAfaAfdT  945 dTUfuGfuUfgGfaGfaGfgGfcUfaCfcdTsdT2319 759 CfuUfuGfaUfgCfcGfgAfcAfaGfcAfdT  946dTGfcUfuGfuCfcGfgCfaUfcAfaAfgdTsdT 2320 760UfuUfgAfuGfcCfgGfaCfaAfgCfcAfdT  947 dTGfgCfuUfgUfcCfgGfcAfuCfaAfadTsdT2321 761 UfuGfaUfgCfcGfgAfcAfaGfcCfaAfdT  948dTUfgGfcUfuGfuCfcGfgCfaUfcAfadTsdT 2322 762GfgAfcAfaGfcCfaCfuGfaGfcGfcAfdT  949 dTGfcGfcUfcAfgUfgGfcUfuGfuCfcdTsdT2323 763 AfcAfaGfcCfaCfuGfaGfcGfcAfaAfdT  950dTUfuGfcGfcUfcAfgUfgGfcUfuGfudTsdT 2324 764GfgAfcUfaCfaGfcCfuGfuCfgUfcAfdT  951 dTGfaCfgAfcAfgGfcUfgUfaGfuCfcdTsdT2325 765 GfaCfuAfcAfgCfcUfgUfcGfuCfaAfdT  952dTUfgAfcGfaCfaGfgCfuGfuAfgUfcdTsdT 2326 766CfuAfcAfgCfcUfgUfcGfuCfaGfcAfdT  953 dTGfcUfgAfcGfaCfaGfgCfuGfuAfgdTsdT2327 767 CfcUfgUfcGfuCfaGfcCfcAfcAfaAfdT  954dTUfuGfuGfgGfcUfgAfcGfaCfaGfgdTsdT 2328 768GfcAfuGfgCfaAfgCfcGfgCfuGfcAfdT  955 dTGfcAfgCfcGfgCfuUfgCfcAfuGfcdTsdT2329 769 CfuGfaCfcAfgAfuAfuGfaCfuGfuAfdT  956dTAfcAfgUfcAfuAfuCfuGfgUfcAfgdTsdT 2330 770GfaUfaUfgAfcUfgUfgAfgGfuGfaAfdT  957 dTUfcAfcCfuCfaCfaGfuCfaUfaUfcdTsdT2331 771 GfgUfgAfaCfgUfgCfcCfgUfgCfuAfdT  958dTAfgCfaCfgGfgCfaCfgUfuCfaCfcdTsdT 2332 772UfaCfaAfgAfuGfgAfcUfuAfcCfuAfdT  959 dTAfgGfuAfaGfuCfcAfuCfuUfgUfadTsdT2333 773 GfgAfcUfuAfcCfuGfgCfaGfaCfuAfdT  960dTAfgUfcUfgCfcAfgGfuAfaGfuCfcdTsdT 2334 774UfuUfuUfcUfgAfgAfuGfcUfcAfcAfdT  961 dTGfuGfaGfcAfuCfuCfaGfaAfaAfadTsdT2335 775 AfgUfaCfaCfaAfuUfgUfuUfuAfcAfdT  962dTGfuAfaAfaCfaAfuUfgUfgUfaCfudTsdT 2336 776AfcAfaGfuUfuGfgUfgCfaUfgUfcAfdT  963 dTGfaCfaUfgCfaCfcAfaAfcUfuGfudTsdT2337 777 AfcUfaAfaAfaGfaUfuCfcUfcGfuAfdT  964dTAfcGfaGfgAfaUfcUfuUfuUfaGfudTsdT 2338 778AfgGfgUfcAfaCfuCfcAfaCfgUfaAfdT  965 dTUfaCfgUfuGfgAfgUfuGfaCfcCfudTsdT2339 779 GfgGfuCfaAfcUfcCfaAfcGfuAfuAfdT  966dTAfuAfcGfuUfgGfaGfuUfgAfcCfcdTsdT 2340 780GfuCfaAfcUfcCfaAfcGfuAfuGfuAfdT  967 dTAfcAfuAfcGfuUfgGfaGfuUfgAfcdTsdT2341 781 UfcAfaCfuCfcAfaCfgUfaUfgUfgAfdT  968dTCfaCfaUfaCfgUfuGfgAfgUfuGfadTsdT 2342 782CfaAfcUfcCfaAfcGfuAfuGfuGfgAfdT  969 dTCfcAfcAfuAfcGfuUfgGfaGfuUfgdTsdT2343 783 CfuCfcAfaCfgUfaUfgUfgGfuUfaAfdT  970dTUfaAfcCfaCfaUfaCfgUfuGfgAfgdTsdT 2344 784UfcCfaAfcGfuAfuGfuGfgUfuAfuAfdT  971 dTAfuAfaCfcAfcAfuAfcGfuUfgGfadTsdT2345 785 CfcAfaCfgUfaUfgUfgGfuUfaUfcAfdT  972dTGfaUfaAfcCfaCfaUfaCfgUfuGfgdTsdT 2346 786AfaCfgUfaUfgUfgGfuUfaUfcUfgAfdT  973 dTCfaGfaUfaAfcCfaCfaUfaCfgUfudTsdT2347 787 UfuAfuAfuCfuGfgGfuUfaAfgUfgAfdT  974dTCfaCfuUfaAfcCfcAfgAfuAfuAfadTsdT 2348 788CfcAfcGfgCfcUfgUfaCfgGfaCfaAfdT  975 dTUfgUfcCfgUfaCfaGfgCfcGfuGfgdTsdT2349 789 AfcGfgCfcUfgUfaCfgGfaCfaCfuAfdT  976dTAfgUfgUfcCfgUfaCfaGfgCfcGfudTsdT 2350 790UfgUfcGfgCfuUfuUfuGfcCfaUfcAfdT  977 dTGfaUfgGfcAfaAfaAfgCfcGfaCfadTsdT2351 791 GfuCfgGfcUfuUfuUfgCfcAfuCfuAfdT  978dTAfgAfuGfgCfaAfaAfaGfcCfgAfcdTsdT 2352 792AfuCfuGfuGfaUfaUfgCfcAfuAfgAfdT  979 dTCfuAfuGfgCfaUfaUfcAfcAfgAfudTsdT2353 793 UfgCfcAfuAfgGfuGfuGfaCfaAfuAfdT  980dTAfuUfgUfcAfcAfcCfuAfuGfgCfadTsdT 2354 794CfcAfuAfgGfuGfuGfaCfaAfuCfcAfdT  981 dTGfgAfuUfgUfcAfcAfcCfuAfuGfgdTsdT2355 795 CfaUfaGfgUfgUfgAfcAfaUfcCfgAfdT  982dTCfgGfaUfuGfuCfaCfaCfcUfaUfgdTsdT 2356 796AfuAfgGfuGfuGfaCfaAfuCfcGfaAfdT  983 dTUfcGfgAfuUfgUfcAfcAfcCfuAfudTsdT2357 797 GfgUfgUfgAfcAfaUfcCfgAfgCfaAfdT  984dTUfgCfuCfgGfaUfuGfuCfaCfaCfcdTsdT 2358 798AfcAfaUfcCfgAfgCfaGfuGfgAfgAfdT  985 dTCfuCfcAfcUfgCfuCfgGfaUfuGfudTsdT2359 799 CfcGfaGfcAfgUfgGfaGfuCfaUfuAfdT  986dTAfaUfgAfcUfcCfaCfuGfcUfcGfgdTsdT 2360 800GfgGfaGfcAfcUfgCfgCfgCfuAfuAfdT  987 dTAfuAfgCfgCfgCfaGfuGfcUfcCfcdTsdT2361 801 GfgAfgCfaCfuGfcGfcGfcUfaUfcAfdT  988dTGfaUfaGfcGfcGfcAfgUfgCfuCfcdTsdT 2362 802AfgCfaCfuGfcGfcGfcUfaUfcCfcAfdT  989 dTGfgGfaUfaGfcGfcGfcAfgUfgCfudTsdT2363 803 UfaUfuGfcUfgCfcAfaGfaGfgGfuAfdT  990dTAfcCfcUfcUfuGfgCfaGfcAfaUfadTsdT 2364 804GfgUfcUfgAfuGfgCfaCfgUfuGfuAfdT  991 dTAfcAfaCfgUfgCfcAfuCfaGfaCfcdTsdT2365 805 CfuGfaUfgGfcAfcGfuUfgUfgGfgAfdT  992dTCfcCfaCfaAfcGfuGfcCfaUfcAfgdTsdT 2366 806GfgCfaCfgUfuGfuGfgGfgUfcGfgAfdT  993 dTCfcGfaCfcCfcAfcAfaCfgUfgCfcdTsdT2367 807 GfcAfcGfuUfgUfgGfgGfuCfgGfgAfdT  994dTCfcCfgAfcCfcCfaCfaAfcGfuGfcdTsdT 2368 808CfaCfgUfuGfuGfgGfgUfcGfgGfgAfdT  995 dTCfcCfcGfaCfcCfcAfcAfaCfgUfgdTsdT2369 809 GfcGfgGfgAfaGfuGfcUfcUfaAfcAfdT  996dTGfuUfaGfaGfcAfcUfuCfcCfcGfcdTsdT 2370 810CfgGfgGfaAfgUfgCfuCfuAfaCfuAfdT  997 dTAfgUfuAfgAfgCfaCfuUfcCfcCfgdTsdT2371 811 UfuAfaGfgUfuUfuGfuUfgCfuAfgAfdT  998dTCfuAfgCfaAfcAfaAfaCfcUfuAfadTsdT 2372 812GfuUfgCfuAfgCfcCfuUfcAfaGfuAfdT  999 dTAfcUfuGfaAfgGfgCfuAfgCfaAfcdTsdT2373 813 GfaGfcUfaUfgUfgAfcUfcGfgAfuAfdT 1000dTAfuCfcGfaGfuCfaCfaUfaGfcUfcdTsdT 2374 814GfcUfaUfgUfgAfcUfcGfgAfuGfgAfdT 1001 dTCfcAfuCfcGfaGfuCfaCfaUfaGfcdTsdT2375 815 CfgGfaUfgGfuCfuUfuCfaCfaCfgAfdT 1002dTCfgUfgUfgAfaAfgAfcCfaUfcCfgdTsdT 2376 816GfaUfgGfuCfuUfuCfaCfaCfgGfcAfdT 1003 dTGfcCfgUfgUfgAfaAfgAfcCfaUfcdTsdT2377 817 UfgGfuCfuUfuCfaCfaCfgGfcAfcAfdT 1004dTGfuGfcCfgUfgUfgAfaAfgAfcCfadTsdT 2378 818AfaCfuAfcCfaUfgAfgAfuGfgUfuAfdT 1005 dTAfaCfcAfuCfuCfaUfgGfuAfgUfudTsdT2379 819 UfaCfcAfuGfaGfaUfgGfuUfuAfgAfdT 1006dTCfuAfaAfcCfaUfcUfcAfuGfgUfadTsdT 2380 820CfcAfaGfcUfcAfcGfaCfcUfuGfgAfdT 1007 dTCfcAfaGfgUfcGfuGfaGfcUfuGfgdTsdT2381 821 AfcGfaCfcUfuGfgAfgCfcCfcGfuAfdT 1008dTAfcGfgGfgCfuCfcAfaGfgUfcGfudTsdT 2382 822GfgGfuAfaGfaGfgGfaCfgAfcAfcAfdT 1009 dTGfuGfuCfgUfcCfcUfcUfuAfcCfcdTsdT2383 823 GfgUfaAfgAfgGfgAfcGfaCfaCfcAfdT 1010dTGfgUfgUfcGfuCfcCfuCfuUfaCfcdTsdT 2384 824GfuAfaGfaGfgGfaCfgAfcAfcCfuAfdT 1011 dTAfgGfuGfuCfgUfcCfcUfcUfuAfcdTsdT2385 825 UfgGfuUfuUfuCfaAfuAfcCfaAfuAfdT 1012dTAfuUfgGfuAfuUfgAfaAfaAfcCfadTsdT 2386 826UfuCfaAfuAfcCfaAfuUfaCfaUfgAfdT 1013 dTCfaUfgUfaAfuUfgGfuAfuUfgAfadTsdT2387 827 AfuAfcCfaAfuUfaCfaUfgGfaAfcAfdT 1014dTGfuUfcCfaUfgUfaAfuUfgGfuAfudTsdT 2388 828CfcAfaCfuAfuUfuAfgUfaAfgCfcAfdT 1015 dTGfgCfuUfaCfuAfaAfuAfgUfuGfgdTsdT2389 829 AfaCfuAfuUfuAfgUfaAfgCfcCfgAfdT 1016dTCfgGfgCfuUfaCfuAfaAfuAfgUfudTsdT 2390 830AfcUfaUfuUfaGfuAfaGfcCfcGfgAfdT 1017 dTCfcGfgGfcUfuAfcUfaAfaUfaGfudTsdT2391 831 AfgAfaAfuUfcCfuUfaGfuCfaUfgAfdT 1018dTCfaUfgAfcUfaAfgGfaAfuUfuCfudTsdT 2392 832CfaUfuAfaGfgGfcAfuUfuUfaCfcAfdT 1019 dTGfgUfaAfaAfuGfcCfcUfuAfaUfgdTsdT2393 833 UfaAfgGfgCfaUfuUfuAfcCfcUfuAfdT 1020dTAfaGfgGfuAfaAfaUfgCfcCfuUfadTsdT 2394 834AfgCfuUfcAfuAfuUfaAfcCfcUfaAfdT 1021 dTUfaGfgGfuUfaAfuAfuGfaAfgCfudTsdT2395 835 UfaUfuAfaCfcCfuAfcCfuGfuCfaAfdT 1022dTUfgAfcAfgGfuAfgGfgUfuAfaUfadTsdT 2396 836UfuAfaCfcCfuAfcCfuGfuCfaAfcAfdT 1023 dTGfuUfgAfcAfgGfuAfgGfgUfuAfadTsdT2397 837 AfcCfcUfaCfcUfgUfcAfaCfgUfaAfdT 1024dTUfaCfgUfuGfaCfaGfgUfaGfgGfudTsdT 2398 838CfcCfuAfcCfuGfuCfaAfcGfuAfaAfdT 1025 dTUfuAfcGfuUfgAfcAfgGfuAfgGfgdTsdT2399 839 CfcUfaCfcUfgUfcAfaCfgUfaAfcAfdT 1026dTGfuUfaCfgUfuGfaCfaGfgUfaGfgdTsdT 2400 840CfuAfcCfuGfuCfaAfcGfuAfaCfgAfdT 1027 dTCfgUfuAfcGfuUfgAfcAfgGfuAfgdTsdT2401 841 UfaCfcUfgUfcAfaCfgUfaAfcGfaAfdT 1028dTUfcGfuUfaCfgUfuGfaCfaGfgUfadTsdT 2402 842AfcCfuGfuCfaAfcGfuAfaCfgAfuAfdT 1029 dTAfuCfgUfuAfcGfuUfgAfcAfgGfudTsdT2403 843 CfcUfgUfcAfaCfgUfaAfcGfaUfuAfdT 1030dTAfaUfcGfuUfaCfgUfuGfaCfaGfgdTsdT 2404 844CfuGfuCfaAfcGfuAfaCfgAfuUfuAfdT 1031 dTAfaAfuCfgUfuAfcGfuUfgAfcAfgdTsdT2405 845 UfgUfcAfaCfgUfaAfcGfaUfuUfcAfdT 1032dTGfaAfaUfcGfuUfaCfgUfuGfaCfadTsdT 2406 846UfcAfaCfgUfaAfcGfaUfuUfcAfuAfdT 1033 dTAfuGfaAfaUfcGfuUfaCfgUfuGfadTsdT2407 847 AfcGfuAfaCfgAfuUfuCfaUfgAfaAfdT 1034dTUfuCfaUfgAfaAfuCfgUfuAfcGfudTsdT 2408 848UfaUfuAfuAfuUfgUfcGfaAfuUfcAfdT 1035 dTGfaAfuUfcGfaCfaAfuAfuAfaUfadTsdT2409 849 UfuAfuAfuUfgUfcGfaAfuUfcCfuAfdT 1036dTAfgGfaAfuUfcGfaCfaAfuAfuAfadTsdT 2410 850UfaUfuGfuCfgAfaUfuCfcUfaCfuAfdT 1037 dTAfgUfaGfgAfaUfuCfgAfcAfaUfadTsdT2411 851 GfaAfuUfcCfuAfcUfgAfcAfaCfaAfdT 1038dTUfgUfuGfuCfaGfuAfgGfaAfuUfcdTsdT 2412 852UfcCfuAfcUfgAfcAfaCfaUfuAfuAfdT 1039 dTAfuAfaUfgUfuGfuCfaGfuAfgGfadTsdT2413 853 UfaUfaAfcUfgUfaUfgGfgAfgCfuAfdT 1040dTAfgCfuCfcCfaUfaCfaGfuUfaUfadTsdT 2414 854UfaAfcUfgUfaUfgGfgAfgCfuUfaAfdT 1041 dTUfaAfgCfuCfcCfaUfaCfaGfuUfadTsdT2415 855 UfgUfaUfgGfgAfgCfuUfaAfcUfuAfdT 1042dTAfaGfuUfaAfgCfuCfcCfaUfaCfadTsdT 2416 856UfuGfaCfaCfuGfgUfaUfcUfuAfuAfdT 1043 dTAfuAfaGfaUfaCfcAfgUfgUfcAfadTsdT2417 857 AfaGfuAfuUfcUfgAfuCfcUfaCfcAfdT 1044dTGfgUfaGfgAfuCfaGfaAfuAfcUfudTsdT

TABLE 5C Efficacy screen results of Hif2α RNAi triggers in viro, asdetermined by dual-luciferase reporter assay. duplex RelativeR_(luc)-Hif2α AD Relative R_(luc)-Hif2α number 1 nM 0.1 nM number 1 nM0.1 nM 2231 0.491 ± 0.198 0.544 ± 0.368 2325 1.379 ± 0.275 1.304 ± 0.1342232 0.468 ± 0.032 0.684 ± 0.061 2326 0.546 ± 0.083 0.660 ± 0.114 22330.862 ± 0.125 0.913 ± 0.019 2327 1.073 ± 0.089 1.339 ± 0.179 2234 0.388± 0.046 0.508 ± 0.148 2328 1.192 ± 0.144 1.291 ± 0.109 2235 0.857 ±0.090 0.743 ± 0.129 2329 0.456 ± 0.064 0.807 ± 0.117 2236 0.761 ± 0.0420.693 ± 0.071 2330 0.464 ± 0.035 1.097 ± 0.416 2237 0.978 ± 0.083 0.746± 0.029 2331 0.783 ± 0.080 1.002 ± 0.321 2238 0.148 ± 0.024 0.208 ±0.023 2332 0.871 ± 0.235 1.174 ± 0.027 2239 0.157 ± 0.017 0.225 ± 0.0222333 0.291 ± 0.019 0.684 ± 0.147 2240 0.845 ± 0.052 0.841 ± 0.048 23340.389 ± 0.044 0.672 ± 0.082 2241 0.270 ± 0.068 0.385 ± 0.028 2335 0.104± 0.013 0.311 ± 0.032 2242 0.222 ± 0.064 0.411 ± 0.019 2336 0.299 ±0.034 0.783 ± 0.037 2243 0.694 ± 0.107 0.688 ± 0.059 2337 0.131 ± 0.0330.334 ± 0.066 2244 0.915 ± 0.115 0.760 ± 0.050 2338 0.334 ± 0.091 0.743± 0.070 2245 0.727 ± 0.066 0.761 ± 0.039 2339 0.189 ± 0.028 0.523 ±0.134 2246 0.327 ± 0.042 0.509 ± 0.044 2340 0.444 ± 0.039 0.920 ± 0.1142247 0.231 ± 0.048 0.439 ± 0.082 2341 0.202 ± 0.018 0.378 ± 0.116 22480.148 ± 0.036 0.215 ± 0.007 2342 0.549 ± 0.041 1.058 ± 0.064 2249 0.190± 0.028 0.303 ± 0.042 2343 0.254 ± 0.009 0.620 ± 0.208 2250 0.139 ±0.046 0.255 ± 0.020 2344 0.276 ± 0.033 0.570 ± 0.071 2251 0.872 ± 0.1210.857 ± 0.087 2345 0.129 ± 0.026 0.296 ± 0.096 2252 0.592 ± 0.061 0.696± 0.096 2346 0.273 ± 0.012 0.523 ± 0.133 2253 0.564 ± 0.043 0.646 ±0.144 2347 0.530 ± 0.040 0.753 ± 0.092 2254 0.641 ± 0.054 0.756 ± 0.0802348 0.454 ± 0.096 0.840 ± 0.270 2255 0.721 ± 0.055 0.653 ± 0.130 23490.504 ± 0.062 0.674 ± 0.172 2256 0.765 ± 0.124 0.796 ± 0.028 2350 0.678± 0.105 0.755 ± 0.085 2257 0.802 ± 0.044 0.647 ± 0.127 2351 1.306 ±0.216 1.216 ± 0.415 2258 0.972 ± 0.168 0.882 ± 0.098 2352 0.749 ± 0.0491.012 ± 0.048 2259 0.836 ± 0.110 0.743 ± 0.086 2353 0.324 ± 0.075 0.635± 0.156 2260 1.030 ± 0.187 0.861 ± 0.031 2354 0.741 ± 0.189 1.043 ±0.239 2261 0.657 ± 0.067 0.568 ± 0.065 2355 0.248 ± 0.063 0.457 ± 0.0662262 0.277 ± 0.071 0.755 ± 0.031 2356 0.210 ± 0.023 0.574 ± 0.006 22630.875 ± 0.059 1.126 ± 0.083 2357 0.435 ± 0.062 0.737 ± 0.082 2264 0.183± 0.019 0.381 ± 0.023 2358 0.731 ± 0.123 0.647 ± 0.113 2265 0.186 ±0.045 0.448 ± 0.042 2359 0.354 ± 0.022 0.576 ± 0.208 2266 0.368 ± 0.0460.808 ± 0.062 2360 0.962 ± 0.167 1.102 ± 0.119 2267 0.651 ± 0.049 1.029± 0.104 2361 0.603 ± 0.133 0.800 ± 0.198 2268 0.210 ± 0.037 0.409 ±0.023 2362 0.454 ± 0.069 0.673 ± 0.089 2269 0.302 ± 0.080 0.530 ± 0.0972363 0.452 ± 0.091 0.653 ± 0.199 2270 0.613 ± 0.147 0.658 ± 0.149 23640.855 ± 0.282 1.070 ± 0.042 2271 0.930 ± 0.094 1.056 ± 0.291 2365 1.259± 0.247 1.132 ± 0.107 2272 0.864 ± 0.197 1.028 ± 0.281 2366 0.695 ±0.115 0.879 ± 0.223 2273 0.588 ± 0.057 0.574 ± 0.033 2367 0.376 ± 0.0390.665 ± 0.064 2274 0.506 ± 0.074 0.870 ± 0.108 2368 1.140 ± 0.221 1.036± 0.063 2275 0.464 ± 0.048 0.515 ± 0.264 2369 0.338 ± 0.055 0.464 ±0.032 2276 0.330 ± 0.080 0.543 ± 0.029 2370 0.294 ± 0.026 0.498 ± 0.1282277 0.702 ± 0.091 0.625 ± 0.384 2371 0.166 ± 0.037 0.193 ± 0.030 22780.564 ± 0.070 0.947 ± 0.044 2372 0.514 ± 0.044 0.831 ± 0.103 2279 0.280± 0.042 0.506 ± 0.086 2373 0.162 ± 0.032 0.369 ± 0.082 2280 0.911 ±0.086 0.871 ± 0.147 2374 0.192 ± 0.014 0.355 ± 0.020 2281 0.651 ± 0.0410.914 ± 0.177 2375 0.305 ± 0.040 0.481 ± 0.062 2282 0.824 ± 0.155 1.209± 0.132 2376 0.228 ± 0.029 0.546 ± 0.115 2283 0.882 ± 0.026 1.175 ±0.194 2377 0.958 ± 0.189 0.900 ± 0.189 2284 0.787 ± 0.062 1.227 ± 0.2662378 0.734 ± 0.170 0.994 ± 0.246 2285 0.723 ± 0.088 1.109 ± 0.247 23790.939 ± 0.204 0.858 ± 0.098 2286 0.878 ± 0.078 1.291 ± 0.367 2380 1.722± 0.101 1.128 ± 0.285 2287 0.751 ± 0.011 0.871 ± 0.102 2381 1.063 ±0.191 0.756 ± 0.109 2288 0.932 ± 0.118 0.943 ± 0.029 2382 1.151 ± 0.3651.070 ± 0.064 2289 1.039 ± 0.146 0.992 ± 0.153 2383 1.060 ± 0.292 0.833± 0.082 2290 0.353 ± 0.036 0.698 ± 0.155 2384 0.439 ± 0.101 0.533 ±0.067 2291 0.434 ± 0.046 0.911 ± 0.092 2385 0.515 ± 0.089 0.701 ± 0.1782292 0.832 ± 0.000 1.135 ± 0.048 2386 0.274 ± 0.087 0.603 ± 0.032 22930.925 ± 0.126 1.010 ± 0.045 2387 0.201 ± 0.063 0.436 ± 0.028 2294 0.546± 0.033 0.743 ± 0.100 2388 0.436 ± 0.021 0.901 ± 0.318 2295 0.863 ±0.122 1.047 ± 0.184 2389 0.820 ± 0.184 1.085 ± 0.307 2296 0.213 ± 0.0280.411 ± 0.079 2390 0.568 ± 0.108 0.772 ± 0.175 2297 0.643 ± 0.028 0.704± 0.083 2391 0.129 ± 0.015 0.273 ± 0.006 2298 0.695 ± 0.037 0.729 ±0.092 2392 0.283 ± 0.052 0.636 ± 0.039 2299 0.827 ± 0.067 0.907 ± 0.1712393 1.049 ± 0.089 0.862 ± 0.388 2300 0.786 ± 0.181 1.090 ± 0.106 23940.254 ± 0.026 0.533 ± 0.076 2301 0.423 ± 0.102 0.558 ± 0.034 2395 0.218± 0.029 0.494 ± 0.104 2302 0.147 ± 0.028 0.414 ± 0.019 2396 0.939 ±0.299 1.321 ± 0.070 2303 0.294 ± 0.034 0.414 ± 0.002 2397 0.119 ± 0.0240.254 ± 0.048 2304 0.302 ± 0.025 0.528 ± 0.116 2398 0.534 ± 0.043 1.047± 0.047 2305 0.992 ± 0.217 0.961 ± 0.120 2399 0.418 ± 0.081 0.784 ±0.071 2306 0.613 ± 0.031 0.596 ± 0.038 2400 0.165 ± 0.020 0.478 ± 0.0282307 0.702 ± 0.142 0.800 ± 0.248 2401 0.174 ± 0.003 0.375 ± 0.062 23080.998 ± 0.059 0.799 ± 0.084 2402 0.128 ± 0.015 0.389 ± 0.060 2309 1.081± 0.135 0.823 ± 0.096 2403 0.568 ± 0.106 0.930 ± 0.132 2310 0.923 ±0.101 0.785 ± 0.122 2404 0.104 ± 0.019 0.234 ± 0.045 2311 0.841 ± 0.1300.888 ± 0.051 2405 0.138 ± 0.035 0.261 ± 0.044 2312 0.725 ± 0.173 0.899± 0.124 2406 0.168 ± 0.044 0.241 ± 0.022 2313 0.886 ± 0.243 0.853 ±0.133 2407 0.124 ± 0.021 0.222 ± 0.027 2314 0.920 ± 0.150 0.692 ± 0.1412408 0.282 ± 0.004 0.730 ± 0.045 2315 0.876 ± 0.099 0.781 ± 0.206 24090.104 ± 0.020 0.301 ± 0.085 2316 0.579 ± 0.055 0.797 ± 0.211 2410 0.154± 0.022 0.228 ± 0.033 2317 0.843 ± 0.157 0.842 ± 0.239 2411 0.410 ±0.083 0.796 ± 0.110 2318 0.780 ± 0.103 0.885 ± 0.370 2412 0.291 ± 0.0140.515 ± 0.037 2319 0.234 ± 0.055 0.454 ± 0.101 2413 0.317 ± 0.055 0.675± 0.104 2320 0.486 ± 0.059 0.770 ± 0.312 2414 0.167 ± 0.038 0.422 ±0.070 2321 0.665 ± 0.033 0.561 ± 0.043 2415 0.119 ± 0.015 0.180 ± 0.0972322 0.306 ± 0.059 0.468 ± 0.012 2416 0.166 ± 0.031 0.247 ± 0.031 23230.664 ± 0.166 0.631 ± 0.085 2417 0.253 ± 0.037 0.295 ± 0.057 2324 1.046± 0.208 1.081 ± 0.293

Example 11. Hif2α RNAi Trigger EC₅₀ Determination

The eight best canonical sequences were further evaluated by determiningthe EC₅₀ concentration. Each trigger was assessed for knockdown underthe same conditions and assays as above, but at 10 differentconcentrations ranging from 0.00051 nM to 10 nM. EC₅₀ were determinedusing GraphPad Prism software. Each of the top five canonical sequenceswere modified to contain UNA at sites 6 and 7. These triggers, alongwith their parent canonical sequences, were evaluated side-by-side forEC₅₀ concentration determination using the same conditions and assays asabove, Table 6.

TABLE 6 EC₅₀ values (nM) determined in vitro for the indicated RNAitriggers. EC50 EC50 UNAs ID number EC50 (nM) ID number Mod. EC50XD-02335 0.0593 AD00988 UNA6 0.8406 AD00993 UNA7 0.5504 XD-02337 0.1010XD-02371 0.0592 AD00989 UNA6 0.3754 AD00994 UNA7 0.08068 XD-02391 0.1554XD-02397 0.0858 XD-02404 0.0570 AD00990 UNA6 0.1534 AD00995 UNA7 0.1689XD-02407 0.0287 AD00991 UNA6 0.3503 AD00996 UNA7 0.1176 XD-02415 0.0892AD00992 UNA6 0.1419 AD00997 UNA7 0.1827

Example 12. Creation of SEAP-Expressing Clear Cell Renal Cell Carcinoma(ccRCC) A498 Cells

A pCR3.1 expression vector expressing the reporter gene secretedalkaline phosphatase (SEAP) under the CMV promoter was prepared bydirectional cloning of the SEAP coding sequence PCR amplified fromClontech's pSEAP2-basic vector. Convenient restriction sites were addedonto primers used to amplify the SEAP coding sequence for cloning intothe pCR3.1 vector (Invitrogen). The resultant construct pCR3-SEAP wasused to create a SEAP-expressing A498 ccRCC cell line. Briefly,pCR3-SEAP plasmid was transfected into A498 ccRCC cells byelectroporation following manufacturer's recommendation. Stabletransfectants were selected by G418 resistance. Selected A498-SEAPclones were evaluated for SEAP expression and integration stability.

Example 13. Orthotopic RCC Tumor Bearing Mice with A498 Cell StablyExpressing SEAP

Female athymic nude mice were anesthetized with ˜3% isoflourane andplaced in the right lateral decubitus position. A small, 0.5-1 cm,longitudinally abdominal incision in the left flank was made. Using amoist cotton swab, the left kidney was lifted out of the peritoneum andgently stabilized. Just before injection, a 1.0 ml syringe was filledwith the cell/Matrigel mixture and a 27 gauge needle catheter wasattached to the syringe tip. The filled syringe was then attached to asyringe pump (Harvard Apparatus, model PHD2000) and primed to removeair. The tip of a 27-gauge needle catheter attached to a syringe wasinserted just below the renal capsule near the caudal pole and the tipof the needle was then carefully advanced cranially along the capsule3-4 mm. A 10 μl aliquot of 2:1 (vol:vol) cell/matrigel mixturecontaining about 300,000 cells was slowly injected into the kidneyparenchyma using a syringe pump. The needle was left in the kidney for15-20 seconds to ensure the injection was complete. The needle was thenremoved from the kidney and a cotton swab was placed over the injectionsite for 30 seconds to prevent leakage of the cells or bleeding. Thekidney was then gently placed back into the abdomen and the abdominalwall was closed. Serum was collected every 7-14 days after implantationto monitor tumor growth using a commercial SEAP assay kit. For moststudies, tumor mice were used 5-6 weeks after implantation, when tumormeasurements were typically around 4-8 mm.

Example 14. Evaluation of HiF2α-RNAi Triggers in Orthotopic RCC TumorBearing Mice

RGD targeted HiF2α-RNAi trigger delivery conjugates. Delivery polymerswere modified using RGD-PEG-HyNic, RGD-PEG-ACit-PNP, orRDG-PEG-FCitFP-TFP and PEG-dipeptide modifying agents. The indicatedamount of polymer 126 or 100A polymer was modified with8×PEG₁₂-ACit-PABC-PNP/0.5× aldehyde-PEG₂₄-FCit-PABC-PNP (with RGD mimic#1-PEG-HyNic using protocol #1) and the indicated amount of theindicated Hif2α RNAi trigger.

Polymer 064 was modified according to protocol 7. Kidney RCCtumor-bearing mice were generated as described and treated with a singletail vein injection of isotonic glucose (G1) or the indicated Hif2α RNAitrigger-delivery polymer conjugate. Mice were euthanized at theindicated time after injection and total RNA was prepared from kidneytumor using Trizol reagent following manufacturer's recommendation.Relative HiF2α mRNA levels were determined by RT-qPCR as described belowand compared to mice treated with delivery buffer (isotonic glucose)only.

TABLE 7 Hif2α knockdown in mice following Hif2α RNAi trigger delivery.RNAi triggers were conjugated to the indicated reversibly modifieddelivery polymer. RNAi trigger Relative Expression duplex Polymer daylow error/ number μg number μg 4 high error isotonic glucose 0 0 1.000.06/0.06 AD01031 80 126 400 0.20 0.04/0.05 AD01214 80 126 400 0.290.08/0.12 AD01214 112.5 006 300 0.36 0.07/0.09 AD01255 80 126 400 0.280.05/0.05 AD01476 115 100A 375 0.32 0.04/0.04 AD01291 80 126 400 0.190.03/0.03 AD01292 80 126 400 0.27 0.06/0.08 AD01293 80 126 400 0.200.01/0.01 AD01294 80 126 400 0.17 0.01/0.02 AD01295 80 126 400 0.220.02/0.02 AD01296 80 126 400 0.21 0.04/0.06 AD01029 80 126 400 0.940.08/0.09 AD01030 80 126 400 0.47 0.08/0.10 AD01256 80 126 400 0.220.05/0.07 AD01257 80 126 400 0.24 0.04/0.05 AD01258 80 126 400 0.380.03/0.03 AD01424 150 100A 300 0.54 0.06/0.07 AD01404 150 100A 300 0.580.11/0.13 AD01405 150 100A 300 0.51 0.10/0.12 AD01406 150 100A 300 0.450.06/0.07 AD01407 150 100A 300 0.47 0.06/0.07 AD01408 150 100A 300 0.500.07/0.09 AD01409 150 100A 300 0.55 0.03/0.03 AD01410 150 100A 300 0.410.10/0.12 AD01411 150 100A 300 0.36 0.01/0.01 AD01288 115 100A 375 0.320.02/0.02 AD01522 115 100A 375 0.44 0.07/0.09 AD01289 115 100A 375 0.280.06/0.08 AD01290 115 100A 375 0.39 0.02/0.02 AD01523 115 100A 375 0.490.04/0.05 AD01524 115 100A 375 0.26 0.05/0.07 AD01554 115 100A 375 0.290.04/0.04 AD01555 115 100A 375 0.20 0.03/0.03 AD01025 80 126 400 0.340.02/0.02 AD01023 80 126 400 0.93 0.10/0.12 AD01024 80 126 400 0.510.04/0.05 AD01028 80 126 400 0.39 0.04/0.04 AD01026 80 126 400 0.970.12/0.13 AD01027 80 126 400 0.92 0.08/0.09 AD01034 80 126 400 0.350.07/0.09 AD01032 80 126 400 1.00 0.09/0.10 AD01033 80 126 400 1.080.08/0.09 AD01022 80 126 400 0.53 0.07/0.09 AD01020 80 126 400 0.940.06/0.06 AD01021 80 126 400 0.90 0.05/0.05 AD01654 100 100A 250 0.1850.01/0.01 AD01655 100 100A 250 0.234 0.02/0.02 AD01656 100 100A 2500.184 0.01/0.01 AD01657 100 100A 250 0.256 0.03/0.03 AD01658 100 100A250 0.138 0.01/0.01 AD01659 100 100A 250 0.249 0.04/0.05 AD01884 100 064250 0.091 0.01/0.01 AD01885 100 064 250 0.146 0.02/0.02 AD01886 100 064250 0.292 0.04/0.05 AD01887 100 064 250 0.329 0.05/0.06 AD01888 100 064250 0.209 0.04/0.08 AD01889 100 064 250 0.282 0.04/0.05 AD01890 100 064250 0.256 0.02/0.03 AD01891 100 064 250 0.189 0.02/0.03 AD01892 100 064250 0.146 0.03/0.03 AD01893 100 064 250 0.115 0.02/0.03 AD02691 75 064187.5 0.124 0.04/0.07 AD02692 75 064 187.5 0.089 0.03/0.04 AD02693 75064 187.5 0.122 0.01/0.01 AD02694 75 064 187.5 0.099 0.01/0.01 AD0269575 064 187.5 0.101 0.02/0.02 AD02733 75 064 187.5 0.283 0.02/0.03AD02734 75 064 187.5 0.262 0.03/0.03 AD02735 75 064 187.5 0.0800.01/0.02 AD01884 75 064 187.5 0.227 0.02/0.02 AD01884 50 064 125 0.2030.03/0.03 AD02692 75 064 187.5 0.121 0.02/0.02 AD02692 50 064 125 0.0920.01/0.01 AD02695 75 064 187.5 0.155 0.02/0.02 AD02695 50 064 125 0.1230.03/0.03 AD02735 75 064 187.5 0.222 0.07/0.10 AD02735 50 064 125 0.1440.03/0.03 AD02857 50 064 125 0.192 0.03/0.03 AD02858 50 064 125 0.1920.04/0.05 AD02859 50 064 125 0.214 0.03/0.03 AD02860 50 064 125 0.1650.02/0.02 AD02949 50 064 125 0.176 0.05/0.07 AD02074 50 064 125 0.5660.01/0.13 AD02861 50 064 125 0.103 0.03/0.05 AD02862 50 064 125 0.0930.05/0.11 AD02873 50 064 125 0.079 0.04/0.06 AD02875 50 064 125 0.1010.01/0.02 AD03011 50 064 125 0.183 0.01/0.02 AD02874 50 064 125 0.1380.05/0.07 AD03187 50 064 125 1.000 0.06/0.07 AD03188 50 064 125 0.3080.08/0.07 AD03189 50 064 125 0.245 0.02/0.02 AD03190 50 064 125 0.2690.08/0.12 AD03191 50 064 125 0.307 0.13/0.24 AD03192 50 064 125 0.2860.01/0.01 AD03193 50 064 125 0.275 0.07/0.09 AD03125 50 064 125 0.2050.04/0.05 AD03126 50 064 125 0.172 0.04/0.04 AD03253 50 064 125 0.1880.04/0.05 AD03264 50 064 125 0.264 0.09/0.13 AD03265 50 064 125 0.2940.07/0.08 AD03266 50 064 125 0.408 0.07/0.08 AD03254 50 064 125 0.2950.06/0.07 AD03255 50 064 125 0.264 0.07/0.10 AD03256 50 064 125 0.3330.06/0.08 AD03257 50 064 125 0.203 0.04/0.05 AD03258 50 064 125 0.1370.02/0.02 AD03259 50 064 125 0.179 0.03/0.04 AD03260 50 064 125 0.1750.03/0.04 AD03261 50 064 125 0.177 0.02/0.02 AD03262 50 064 125 0.1970.01/0.01 AD03263 50 064 125 0.134 0.04/0.06

Quantitative Real-Time PCR assay. In preparation for quantitative PCR,total RNA was isolated from tissue samples homogenized in TrReagent(Molecular Research Center, Cincinnati, Ohio) following themanufacturers protocol. Approximately 500 ng RNA was reverse-transcribedusing the High Capacity cDNA Reverse Transcription Kit (LifeTechnologies). For human (tumor) Hif2α (EPAS 1) expression,pre-manufactured TaqMan gene expression assays for human Hif2α (Catalog#4331182) and CycA (PPIA) Catalog #: 4326316E) were used in biplexreactions in triplicate using TaMan Gene Expression Master Mix (LifeTechnologies) or VeriQuest Probe Master Mix (Affymetrix). For human(tumor) VegFa (VEGFA) expression, pre-manufactured TaqMan geneexpression assays for human VegFa (Catalog #4331182, Assay ID:Hs00900055) and CycA (Part #: 4326316E) were used in biplex reactions intriplicate using TaqMan Gene Expression Master Mix (Life Technologies)or VeriQuest Probe Master Mix (Affymetrix). Quantitative PCR wasperformed by using a 7500 Fast or StepOnePlus Real-Time PCR system (LifeTechnologies). The AACT method was used to calculate relative geneexpression.

Example 15. Multi-Dose Hif2α RNAi Trigger-Delivery Polymer ConjugateInhibits Tumor Growth in Orthotopic RCC Tumor Bearing Mice

Hif2α RNAi trigger-delivery polymer conjugate was prepared usingprotocol #1 with RNAi trigger duplex ID AD01031 and polymer Ant 126. Theconjugate was then TFF purified and polymer concentration, RNAi trigger,RGD and modifying conjugation efficiency was determined as describedabove. Weekly doses of Hif2α RNAi trigger-delivery polymer conjugatecontaining either 400 μg (polymer weight) or 280 μg (polymer weight)were administered intravenously to 2 different groups of tumor bearingmice. Tumor bearing mice receiving isotonic glucose (IG) were used astreatment control. A total of 3 weekly doses were administered duringthe course of study. Tumor growth rates were evaluated by serum SEAPcollected at 5-7 days interval during treatment. Tumor weight and volumewas determined at necropsy. Gross tumor morphology and H&Ehistopathology were evaluated.

TABLE 8 Hif2α RNAi trigger-delivery polymer conjugate knockdown HiF2αand VEGFa Hif2α expression VEGFa expression Treat- Relative low/highRelative low/high ment Expression error Expression error G1-IG 1.000.07/0.07 1.00 0.07/0.08 G2-400 μg 0.18 0.03/0.04 0.45 0.07/0.09 G3-280μg 0.19 0.04/0.05 0.39 0.10/0.13

Expression of Hif2α in the 400 μg or 280 μg Hif2α RNAi trigger-deliverypolymer conjugate group was 82% and 81% decreased, respectively,compared to control treatment (Table 8). Expression of VEGFa, a wellcharacterized down-stream Hif2α regulated gene, was also decreased by55% and 61%, respectively (Table 8).

Collectively, after 3 weekly Hif2α RNAi trigger-delivery polymerconjugate injections, tumor growth was dramatically inhibited in bothdosages evaluated. This is supported by the overall tumor sizes andserum SEAP levels (FIGS. 8-9 and 12, Table 9). A downward trend of theSEAP levels after the third injections suggest beginning of tumorregression. In addition, tumor histopathology examination from H&Estained formalin fixed paraffin sections showed destruction of typicalRCC tubular structure. The number of apoptotic cells was increased intreatment groups. Some tumor sample contained large areas of tumornecrosis (FIG. 10).

TABLE 9 Serum SEAP levels during treatment, G1 and G2, n = 4; G3, n = 3SEAP (fold-changed relative to day −1) Treatment Day −1 Day 7 Day 14 Day21 G1-IG 1.00 ± 0.19 1.65 ± 0.22 3.44 ± 0.23 7.48 ± 0.27 G2-400 μg 1.00± 0.45 0.79 ± 0.43 1.36 ± 0.61 1.25 ± 0.68 G3-280 μg 1.00 ± 0.14 1.46 ±0.09 2.55 ± 0.29 1.82 ± 0.35

Example 16. Evaluation of HiF2α-RNAi triggers in orthotopic RCC tumorbearing mice. RGD targeted HiF2α-RNAi trigger delivery polymerconjugates were formed using polymer 126, 100A, or 006. The RNAitrigger, μg indicates the quantity of trigger reacted with polymer. Thepolymer was modified with the indicated RGD mimic and PEG modifyingagents as described above. Kidney RCC tumor-bearing mice were generatedas described and treated with a single tail vein injection of isotonicglucose (G1) or the indicated Hif2α RNAi trigger-delivery polymerconjugate. Mice were euthanized 72 h (day 4) after injection and totalRNA was prepared from kidney tumor using Trizol reagent followingmanufacturer's recommendation. Relative HiF2α mRNA levels weredetermined by RT-qPCR as described and compared to mice treated withdelivery buffer (isotonic glucose) only (Table 11).

Example 17. HiF2α RNAi Trigger/Second Therapeutic Combination Study

HiF2α RNAi trigger-delivery polymer conjugate (125 μg polymer) wasprepared using protocol 7 Duplex ID No. AD1884 and polymer 064. HiF2αRNAi trigger-delivery polymer conjugate was dosed every 4 weeks by ivinjection, 4 doses total. Sunitinib (Malate salt) obtained from LClaboratories was suspended in Ora-plus/Ora sweet (50:50, vol:vol).Sunitinib treatment started 2 weeks after the first HiF2α RNAi triggerdose was administered. Mice were dosed by oral gavage 5 days/week for 2weeks, then off 2 weeks, 3 cycles total.

Tumor growth rates were evaluated by serum SEAP collected at 5-7 daysinterval during treatment. Tumor weight and volume was determined atnecropsy. Gross tumor morphology and H&E histopathology were evaluated.Relative HiF2a expression levels were of were 11.4%, 73.8%, and 77.6%decreased in the sunitinib alone, DPC+sunitinib and DPC alone treatedgroups, respectively (Table 10A). Combined HiF2a RNAi trigger andsunitinib treatments resulted in increased tumor growth inhibitionresponse. Overall smaller tumor sizes were smaller and lower overallgrowth (as measured by overall-fold increase in SEAP) was observed(Table 10B).

TABLE 10A HiF2α expression in RCC tumors in animal models treated withSunitinib, HiF2α RNAi trigger, or HiF2α RNAi trigger + Sunitinib. HiF2αexpression Treatment Relative expression Low error High error isotonicglucose 1.000 0.153 0.180 Sunitinib 0.886 0.070 0.076 HiF2α RNAi trigger0.262 0.071 0.097 HiF2α RNAi trigger + 0.224 0.081 0.126 Sunitinib

TABLE 10B Tumor size and SEAP expression in RCC tumors in animal modelstreated with Sunitinib, HiF2α RNAi trigger, or HiF2α RNAi trigger +Sunitinib. Treatment Tumor weight (mg) Tumor volume (mm3) Fold increasein SEAP isotonic glucose 4158.5 ± 865  3576.3 ± 279  21.8 ± 11 Sunitinib2385.7 ± 845  2113.5 ± 368   18.1 ± 9.8 HiF2α RNAi trigger 2130.9 ± 10661537.4 ± 999 20.54 ± 20 HiF2α RNAi trigger + 1075.8 ± 600  1008.8 ± 650  5.7 ± 3.3 Sunitinib

Other Embodiments: It is to be understood that while the invention hasbeen described in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

TABLE 11 Hif2α expression in RCC cells in RCC tumor bearing micefollowing Hif2α RNAi trigger delivery. HiF2α polymer Relative ExpressionRNAi trigger purifi- RGD PEG error duplex ID μg ID μg cation modifyingagent amt. no. modifying agent day 4 low high isotonic glucose 1.0000.060 0.064 AD01031 80 126 280 RGD-PEG₈-HyNic 0.5× PEG₁₂-ACit-PABC-PNP0.300 0.074 0.098 AD01031 80 126 280 RGD-PEG₈-ACit- 0.4×PEG₆-ACit-PABC-PNP 0.311 0.055 0.067 PABC-PNP AD01214 80 126 280 TFFRGD-PEG₈-HyNic 0.5× PEG₁₂-FCit-PABC-PNP 0.489 0.022 0.023 AD01214 80 126280 RGD-PEG₈-HyNic 0.5× PEG₁₂-FCitFP-TFP 0.210 0.032 0.038 AD01214 80126 280 RGD-PEG₈-ACit- 0.4× PEG₆-ACit-PABC-PNP 0.360 0.019 0.021PABC-PNP AD01214 80 126 280 RGD-PEG₈-ACitFP-NHS   1× PEG₆-ACit-PABC-PNP0.288 0.005 0.005 AD01214 115 100A 375 RGD-PEG₈-HyNic 0.5×PEG₁₂-FCitFP-TFP 0.258 0.033 0.038 AD01214 112.5 100A 375 TFFRGD-PEG₁₅-FCitFP-TFP 0.5× 10.8 PEG₁₂-ACit-PABC-PNP 0.193 0.046 0.061AD01214 112.5 100A 375 TFF RGD-PEG₁₅-FCitFP-TFP   1× 16.1PEG₁₂-ACit-PABC-PNP 0.182 0.007 0.008 AD01214 112.5 100A 375 TFFRGD-PEG₁₅-FCitFP-TFP   2× 29.0 PEG₁₂-ACit-PABC-PNP 0.182 0.031 0.038AD01214 112.5 100A 375 TFF RGD-PEG₁₉-FCitFP-TFP 0.5× 10.7PEG₁₂-ACit-PABC-PNP 0.163 0.023 0.027 AD01214 112.5 100A 375 TFFRGD-PEG₁₉-FCitFP-TFP   1× 18.5 PEG₁₂-ACit-PABC-PNP 0.114 0.011 0.012AD01214 112.5 100A 375 TFF RGD-PEG₁₉-FCitFP-TFP   2× 31.1PEG₁₂-ACit-PABC-PNP 0.182 0.047 0.063 AD01214 112.5 100A 375RGD-PEG₁₉-FCitFP-TFP   1× PEG₁₂-FCitFP-TFP 0.148 0.079 0.169 AD01214112.5 100A 375 RGD-PEG₁₉-FCitFP-TFP   1× PEG₁₂-FCitFP-TFP 0.188 0.0260.03 AD01214 112.5 100A 375 RGD-PEG₁₉-FCitFP-TFP   1×PEG₁₂-ACit-PABC-PNP 0.195 0.043 0.055 AD01214 112.5 006 300RGD-PEG₈-HyNic PEG₁₂-ACit-PABC-PNP 0.357 0.069 0.086 AD01214 112.5 100A375 TFF RGD-PEG₂₀-FCitFP-TFP 0.125×  1.9 PEG₁₂-ACit-PABC-PNP 0.169 0.0520.075 AD01214 112.5 100A 375 TFF RGD-PEG₂₀-FCitFP-TFP 0.25×  3.4PEG₁₂-ACit-PABC-PNP 0.168 0.029 0.035 AD01214 112.5 100A 375 TFFRGD-PEG₂₀-FCitFP-TFP 0.5× 6.6 PEG₁₂-ACit-PABC-PNP 0.130 0.004 0.005AD01214 112.5 100A 375 TFF RGD-PEG₂₀-FCitFP-TFP   1× 12.7PEG₁₂-ACit-PABC-PNP 0.121 0.016 0.018 AD01214 112.5 100A 375 TFFRGD-PEG₂₀-FCitFP-TFP 1.5× 20.3 PEG₁₂-ACit-PABC-PNP 0.135 0.018 0.020

1. A composition comprising an RNA interference (RNAi) trigger forinhibiting the expression of an Hif2α gene, wherein the RNAi triggercomprises a sense strand and an antisense strand, wherein said antisensestrand comprises the base sequence of nucleotides 2-19 of any of theantisense strands of Table 1, Table 2 or Table
 5. 2. The composition ofclaim 1, wherein the antisense strand comprises a nucleotide basesequence of nucleotides 2-21 of SEQ ID NO. 4 (UUCAUGAAAUCGUUACGUUG) orSEQ ID NO. 38 (GUAAAACAAUUGUGUACUUU).
 3. The composition of claim 2,wherein a) the antisense strand comprises a nucleotide base sequence ofnucleotides 2-21 of SEQ ID NO. 4 (UUCAUGAAAUCGUUACGUUG) and the sensestrand comprises a nucleotide base sequence of SEQ ID NO. 53(ACGUAACGAUUUCAUGAAU), or b) the antisense strand comprises a nucleotidebase sequence of nucleotides SEQ ID NO. 38 (GUAAAACAAUUGUGUACUUU) andthe sense strand comprises a nucleotide base sequence of nucleotides1-19 of SEQ ID NO. 56 (AGUACACAAUUGUUUUACA).
 4. The composition of claim1, wherein the sense strand and/or the antisense strand furthercomprises a 3′ and/or 5′ extension of 1-6 nucleotides in length.
 5. Thecomposition of claim 1, wherein a targeting group is conjugated to theRNAi trigger.
 6. The composition of claim 5, wherein the targeting groupcomprises a compound selected from the group consisting of:integrin-binding compound, α_(v)β₃ integrin-binding ligand, RGD peptideligand, and RGD mimic.
 7. The composition of claim 1, wherein a deliverypolymer is conjugated to the RNAi trigger.
 8. The composition of claim1, wherein a linking group is conjugated to the RNAi trigger.
 9. Thecomposition of claim 1, wherein the sense strand and/or antisense strandindependently comprises one or more modified nucleotides or nucleotidemimics.
 10. The composition of claim 9, wherein the sense strandcontains one, two, or three 2′-deoxy-2′-fluoro modified nucleotides atpositions 11, 12, and/or 13 from the 3′ end.
 11. The composition ofclaim 9, wherein the antisense strand contains a 2′-deoxy-2′-fluoromodified nucleotide at position 2 from the 5′ end.
 12. The compositionof claim 9, wherein the antisense strand contains a 2′-deoxy-2′-fluoromodified nucleotide at position 14 from the 5′ end.
 13. The compositionof claim 9, wherein the antisense strand contains one, two, three, orfour 2′-F nucleotides at positions 4, 6, 8, 10, and 12 from the 5′ end.14. The composition of claim 1, wherein the RNAi trigger comprises oneor more phosphorothioate internucleotide linkages.
 15. The compositionof claim 14, wherein the antisense strand contains one, two, three, orfour phosphorothioate internucleotide linkages.
 16. The composition ofclaim 1, further comprising one or more additional therapeutics ortreatments.
 17. The composition of claim 1 further comprising apharmaceutically acceptable excipient.
 18. The composition of claim 1,wherein said composition is packaged in a kit, container, pack,dispenser, pre-filled syringes, or vials.
 19. A method for inhibitingHif2α expression in a cell, tissue, or subject, the method comprising:administering to the subject a therapeutically effective amount of acomposition of claim
 1. 20. The method of claim 19, wherein thecomposition is administered via subcutaneous injection.
 21. The methodof claim 19, wherein the cell or tissue is a renal cell carcinoma cell.22. An (RNAi) trigger capable of inhibiting the expression of an Hif2αgene, wherein the RNAi trigger comprises a sense strand and an antisensestrand, wherein said antisense strand comprises any of the antisensesequences in Table 1, Table 2, or Table 5.